fredag 3 januari 2025

Uppslagsförfrågan (PHI)

Alla helger börjar nu gå mot sitt slut och de sista resterna av jul & nyårsmaten är snart uppätna (undertecknad är vansinnigt förtjust i inläggningar och det som förknippas med jul och nyårsmaten) så mitt lystmäte har verkligen fått sitt. Men efter trettondagshelgen får det vara nog med att smörja kråset och istället gå in i vardagen. 

Så min fråga till er läsare är: Vad vill ni läsa om här på bloggen 2025? Vilka ämnen/områden är mest intressanta? 
Vad har jag missat som är värt att belysas?
Utöver det jag vanligtvis bevakar och skriver om kan det vara kul att få input om uppslag till nya framtida inlägg.
Sååå..hit me with your best uppslag.

                                            Mvh the99

söndag 29 december 2024

Mellandagsinlägg - HoloMonitor vid läkemedelsforskning

Halloj alla phi,are. Hoppas ni haft en bra jul med allt det innebär.Snart glider vi in i ett nytt år så undertecknad börjar så smått värma upp motorerna. Här kommer en studie daterad 23/12 2024 som tar upp tekniken vid framtagning av nya läkemedel. Ett gäng forskare från Italien,Danmark och Belgien har studerat hur förbättra forskning och utveckling av nya läkemedel.Man har fokuserat på 2 beståndsdelar som ofta förekommer i cancerläkemedel,nocodazole och paclitaxel.Hur snabbare och effektivare få fram kommande läkemedel. Genom studiens utförande har de haft "kontrollstationer" för att se läkemedlens effektivitet in action på människoceller. PHI`s excellenta HoloMonitor har använts för det syftet.

Forskarna utgår från det faktumet att utveckling av nya läkemedel kostar multum (miljardbelopp) och inte sällan desssvärre inte når hela vägen fram.Ett misslyckande kan alltså slå hårt på även de allra största bjässarna i branschen. De av er som varit med på svenska biotechföretag som sysslar med forskning och gått på pumpen vet konsekvenserna. Därför är behovet av kontrollstationer tidigt in i processen något som kommer gå upp för fler och fler biotechföretag. Men till studien: 

HYDRA: HYdrogel Dispensing with Robotic Automation for high throughput drug testing Posted December 23,2024

Abstract

The poor predictivity of traditional cell culture platforms hampers drug research and development efficiency................

1 Introduction

Predicting how human cells will respond to new therapeutics accurately in preclinical stages is essential for enhancing drug research and development (R&D). However, this process has become notably lengthy, resource-intensive, and risky: typical efforts last ten years, cost over 1 billion US$, and fail nine out of ten times. Genomics and artificial intelligence advancements have led to improvements,but R&D still faces declining productivity and high failure rates in clinical trials.It has been estimated that up to 50% of potential therapeutics that succeed in the initial preclinical assays fail in the following clinical trials, suggesting that existing preclinical models have a low capacity to predict human responses.For example, drug development has relied on cell culture assay using plastic or glass substrates for decades. However, these stiff and inert substrates lack many physicochemical characteristics of soft and active biological tissues. These differences lead to loss of tissue-specific architecture, altered mechanical and metabolic signals, and distinct interactions between cells and their extracellular matrix (ECM). Together, these differences make traditional cell culture platforms poor preclinical models.

Studiens HoloMonitoravsnitt

Drug screening using digital holographic long-term imaging

Nocodazole (M1404-50MG, Merck) and paclitaxel (10461, Cayman Chemical) were chosen as test compounds. The drugs were dissolved in dimethylsulphoxide (D2650-100ML, Merck), aliquoted in 1 mL Eppendorf vial, and kept at −20 °C. 10% w/v FG-based culture platform was fabricated as described previously (see “Preparation of transglutaminase-crosslinked fish gelatin hydrogels” and “Automated hydrogel fabrication using liquid handling robots”). HaCaT cells (P50-P70, 50.000 cells/mL) were seeded on 10% w/v FG hydrogels 24 h before treatment by casting 100 μL per well of the 96-well plate. In the drug screening, cells were treated with three different concentrations per drug, and positive and negative controls (resulting in 12 replicates per group) were included. Specifically, nocodazole and paclitaxel (5 mg/mL) were diluted to 5 μg mL−1 solutions in a DMEM culture medium. Cells were treated with nocodazole concentrations of 12.5, 25, and 50 ng mL−1 and paclitaxel concentrations of 0.5, 2.5, and 12.5 ng mL−1. Wells with 0.1% DMSO and 1500 µg mL−1 Geneticin (10131-027, Gibco) were used as a negative and positive control, respectively. After adding drugs, the 96-well plate was immediately placed on the motorized stage of a HoloMonitor M4 (Phase Holographic Imaging AB) inside a cell incubator (37 °C, 100% humidity, 5% CO2). Using HoloMonitor Appsuite software (Version 4.0.1, Phase Holographic Imaging PHI AB), one field of view per well was selected and imaged every hour for 48 hours using an Olympus PLN 20X objective (NA 0.4) (N1215900, Evident) and a low-power laser unit (635 nm, 0.2 mW cm2). Time-lapse images were exported and processed on ImageJ FIJI software to extract the cell % confluency. In detail, the cell confluency was defined as the sum of areas occupied by cells / the total size of the field of view) (Figure 4, S6, S7, S8, and Videos s5, s6).

Cell confluency analysis using digital holographic long-term imaging

To extract the cell confluency value (defined as the percentage of the field of view featuring cells), time-lapse datasets from the HoloMonitor microscope were exported from HoloMonitor Appsuite software (Version 4.0.1, Phase Holographic Imaging PHI AB) and processed using the open-source ImageJ FIJI software.

Drug test using phase holographic imaging.

A) Holographic image of HaCaT cultured in 0.1% DMSO – vehicle control – after seeding and B) 48 hours after seeding. C) Holographic image of HaCaT treated with 50 ng mL−1 of nocodazole drug 48 hours after seeding. Scale bars: 50 µm. D) HaCaT cell confluency vs. time. Cells were cultured in nocodazole-based cell culture media (12.5 ng/mL – blue dots, 25 ng/mL – red squares, 50 ng/mL black triangles) for 48 hours on hydrogel substrates. Data were normalized concerning the initial confluency value. Solid lines represent mean values, and shaded areas represent s.e.m (n=12). E) Final cell confluency vs. nocodazole concentration (12.5 ng/mL, 25 ng/mL, 50 ng/mL) on plastic and hydrogel substrates. Data were normalized concerning the initial confluency value. (*) stands for significative difference. F) Nocodazole dose-response curves on plastic (IC50, 16.6 ng mL−1) and hydrogel (IC50, 18.1 ng mL−1) substrates (n=12). Data were normalized concerning the initial confluency value (n=12). Image brightness and contrast were adjusted for printed visibility.

F) Time-lapse holographic images of HaCaT cells on hydrogel substrates acquired during 48 hours of treatment with nocodazole or paclitaxel. Scale bar: 50 μm.


Min kommentar


PHI med sin teknik manifisterad i HoloMonitor ligger alltsomoftast före sin tid har det visat sig. 
Månne det nu är skördetid för instrumentet inom läkemedelsforskningen? Länk

                                         Mvh the99

Som service till alla HoloMonitornyfikna forskare : HoloMonitor Demo

tisdag 24 december 2024

God Jul!

Vill bloggen önska alla hårt prövade phi,are. Glöm börsen och allt vad aktier heter nu under juldagarna och ägna er istället åt god julmat & trevligt umgänge. Mvh the99

måndag 23 december 2024

PHI - PM

Phase Holographic Imaging PHI
PHI announces outcome of rights issue (Cision)

2024-12-23 17:19

PHI announces outcome of rights issue

NOT FOR RELEASE, DISTRIBUTION OR PUBLICATION, DIRECTLY OR INDIRECTLY, IN THE UNITED STATES, AUSTRALIA, JAPAN, CANADA, NEW ZEALAND, SOUTH AFRICA, HONG KONG, SWITZERLAND, SINGAPORE, SOUTH KOREA, RUSSIA, BELARUS OR IN ANY OTHER JURISDICTION WHERE THE RELEASE, DISTRIBUTION OR PUBLICATION OF THIS PRESS RELEASE WOULD BE UNLAWFUL OR REQUIRE ADDITIONAL REGISTRATION OR OTHER MEASURES BEYOND THOSE REQUIRED BY SWEDISH LAW. PLEASE SEE "IMPORTANT INFORMATION" AT THE END OF THIS PRESS RELEASE.

The subscription period in Phase Holographic Imaging PHI AB’s ("PHI" or the "Company") rights issue of shares of a total of SEK 64.3 million ended on 20 December 2024 (the "Rights Issue"). The Rights Issue was subscribed to a total of approximately 51.5 percent, including the pre-subscription commitment from Altium SA, meaning that top and bottom underwriting commitments of a total of approximately 48.5 percent are activated to achieve a total subscription rate of 100 percent. PHI will thus be provided proceeds of approximately SEK 64.3 million before the deduction of transaction related costs and offsetting of loans.

Goran Dubravčić, Chairman of PHI and CEO of Altium, comments

“Having been part of the capital raising for PHI in the Swedish market has been an exciting experience for me and Altium. Through the rights issue, Altium has become an even larger shareholder, and we stand by our commitment to support PHI, that now has the resources to deliver on set objectives, which is important in the collaboration between Altium and PHI”, says Goran Dubravčić, Chairman of PHI and CEO of Altium.

Anders Månsson, new CEO of PHI, comments

“In this challenging market, I am humbled by the commitment by the shareholders and new investors in PHI shown through the rights issue. Thank you! As I have mentioned throughout the capital raising period, the rights issue is a strategic move to not only strengthen our financial position but also ensure the resources needed to take the next steps in our development. Now we have the funds to accelerate our growth and advance to the next phase of our development”, says Anders Månsson, CEO of PHI.

Subscription, allocation and payment

The Rights Issue was subscribed to approximately SEK 33.1 million including pre-subscription commitment from Altium SA of approximately SEK 15.2 million, which corresponds to a subscription rate of approximately 51.5 percent. In addition, top and bottom underwriting commitments of a total of approximately SEK 31.2 million have been activated, corresponding to approximately 48.5 percent of the Rights Issue. The activated underwriting commitments refer with approximately SEK 10.7 million to the bottom underwriting commitment and approximately SEK 20.5 million to the top underwriting commitment. The total subscription rate (including underwriting commitments) thus ended at 100 percent, meaning that PHI is provided with approximately SEK 64.3 million before transaction related costs of approximately SEK 9.6 million (of which approximately SEK 5.3 million refers to underwriting fees, provided that all underwriters desire to receive cash compensation) and offsetting of loans.

A total of 18,254,089 shares were subscribed for with the support of pre-emptive subscription rights, including pre-subscription commitments. In total, 17,475,253 shares were subscribed without the support of pre-emptive subscription rights, including activated underwriting commitments. Through the Rights Issue, 35,729,342 new shares will be issued. The subscribers who are allocated shares without preferential rights will receive settlement notes, which are planned to be sent out after a formal board decision on allocation. Allotted shares without pre-emptive rights shall be paid in accordance with the instructions on the settlement note.

Compensation for underwriting commitments

For the bottom underwriting commitment, the underwriters receive an underwriting compensation of 10 percent in cash or 13 percent in the form of shares (at the same terms as in the Rights Issue). For the top underwriting commitment, the underwriters receive an underwriting compensation of 12 percent in cash or 15 percent in the form of shares (at the same terms as in the Rights Issue).

Number of shares and share capital

When the Rights Issue has been registered with the Swedish Companies Registration Office, the total number of shares will have increased by 35,729,342 shares, from 27,322,438 shares to 63,051,780 shares and the Company's share capital will, upon registration, have increased by SEK 7,145,868.40, from SEK 5,464,487.60 to SEK 12,610,356.00. The shareholders who have not participated in the Rights Issue will be subject to a dilution effect corresponding to approximately 57 percent of the votes and capital.

Trading in BTA

Trading in BTA's will take place on Spotlight Stock Market until the Rights Issue has been registered with the Swedish Companies Registration Office. The registration is expected to take place around 13 January 2025.

Flagging

After the Rights Issue, Altium SA has passed the flagging limit of 30 percent. Before the Rights Issue, Altium SA’s holding corresponded to approximately 23.6 percent of votes and capital in the Company. After the Rights Issue, Altium SA’s ownership corresponds to approximately 41.6 percent of votes and capital in the Company. Altium SA, in advance, as previously communicated, has by the Swedish Securities Council (Swe: Aktiemarknadsnämnden) been granted exemption from the mandatory bid requirements if its shareholdings would amount to or exceed 30 percent of the number of votes in the Company because of Altium’s participation in the Rights Issue.

Lock up

Altium has, through a lock-up agreement, with Navia Corporate Finance AB as counterparty, undertaken, with customary exceptions, not to sell any of the existing shareholding or such shareholding that has been acquired through subscription in the Rights Issue, for a period of six months after the completion of the Rights Issue. 

Medicinteknikbolaget Phase Holographics företrädesemission tecknades till 51,5 procent med stöd av teckningsrätter. Emissionsgaranten Altium tecknar resterande 48,5 procent och företrädesemissionen blir därmed fulltecknad. Bolaget tillförs 64,3 miljoner kronor före emissionskostnader på 9,6 miljoner kronor och kvittning av lån. Det framgår av ett pressmeddelande.

Bolaget emitterar 35,73 miljoner nya aktier och har därefter 63,05 miljoner aktier utestående.

Altium ökar sitt ägande till 41,6 procent av aktierna i bolaget.

söndag 22 december 2024

6 nya forskningsrapporter (2 extraordinära)

1. I ett samarbetsprojekt har 14 forskare från Frankrike,Kina och Australien fått sina studier publicerade om ett ämne jag inte är närmare bekant med,nämligen hydrogeler inom Reg Med som material att använda vid 3D vävnadstillverkning.

High-Performance Sunlight-Induced Polymerized Hydrogels and Applications in 3D and 4D Printing 18 Dec 2024

Abstract

Currently, there are only few reports on water-soluble photoinitiating systems. In this study, a highly water-soluble organic dye i.e. sodium (E)-3,3′-((4-(2-(3-methylbenzo[d]thiazol-3-ium-2-yl)vinyl)phenyl)azanediyl)dipropionate iodide, was synthesized and served as a photoinitiator. Notably, this water-soluble initiator, at a low concentration of just 0.01 wt%, demonstrates a high photoinitiation ability, with some hydrogel formulations achieving nearly 100% double bond conversion under sunlight. Photopolymerization kinetics were monitored using Real-Time Fourier Transform Infrared. To explore the complex chemical principles of radical polymerization, UV-visible absorption and fluorescence spectroscopy, steady-state photolysis, fluorescence quenching experiments and cyclic voltammetry were employed to gain a comprehensive understanding of the photochemical mechanism involved. Additionally, several characteristics of the synthesized hydrogels were also investigated i.e. the water content, the water swelling, and the volume swelling. In addition to their excellent photoinitiation capabilities, the hydrogel formulations developed in this study also supported 3D printing. 3D objects with smooth surface and a high spatial resolution could be successfully printed using direct laser writing. The fabricated hydrogels could reversibly change of shape in response to water (adding or removing water), enabling successful 4D printing behavior. Furthermore, the efficient photoinitiation ability of the water-soluble formulations opens new avenues for sunlight-polymerized hydrogels and potential applications in bioprinting.

2.2 Study on Cytotoxicity of Dye-A1

It can be seen that the cells have already died, with only few cells remaining visible under the Holomonitor (see Figure S2, Supporting Information). As shown in Figure S3 (Supporting Information), the track of single cell movement in the culture medium containing dye-A1 shows that the cells had vitality, and the number of cells was significantly higher than that of the control TPO (see Figure S4, Supporting Information). The above results show that dye-A1 did not cause any cytotoxicity. Therefore, dye-A1 can support the growth, proliferation, and survival of cells, demonstrating excellent biocompatibility.



Figure S4. The Holomonitor images of dye-A1. 

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2. 6 forskare från Taiwan har fått sina studier om neutrofila granulocyter,som är vita blodkroppar och ingår i kroppens immunsystem,publicerade. Studien är dessvärre hårt inlåst men Google Scholar har lagt den under Studier med HoloMonitor så vi får lita på dem. Chang Gung är ett HoloMonitoruniversitet så det vidimerar påståendet.

Myriscagayanone C, a new compound from the fruit of myristica cagayanensis, inhibits fMLP-induced respiratory bursts by specifically preventing Akt translocation in human neutrophils 17 Dec 2024

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3. 4 Forskare från Grekland har fått sina studier om probiotika (mölksyrebakterier) kontra celldöd publicerade. Noterbart att Grekland inte finns upptagna under PHI`s User-lista så det bör vara en ny kund.

Lactobacilli-host interactions inhibit Staphylococcus aureus and Escherichia coli-induced cell death and invasion in a cellular model of infection 18 Dec 2024

Probiotic lactobacilli exert antimicrobial and antibiofilm activity through a plethora of mechanisms, including the production of inhibitory compounds and competition for nutrients and adhesion sites on the host mucosa. Their ability to interfere with pathogen-host interactions may mitigate the harmful effects of infection. Lactiplantibacillus pentosus L33 (L33) and Lactiplantibacillus plantarum L125 (L125) are two potential probiotic lactic acid bacteria (LAB) strains, previously shown to reduce pathogen viability and biofilm formation in vitro. The present study aims to investigate their ability to limit cell death induced by Staphylococcus aureus and Escherichia coli in the human colon adenocarcinoma cell line HT-29. To this end, we examined the protective effects of the two strains using in silicoin vitro and omic approaches, with Lacticaseibacillus rhamnosus GG (LGG) serving as a reference strain, due to its well-documented antimicrobial properties. Based on the findings of our study, direct contact of HT-29 cells with L125 for 4 h prior to the addition of S. aureus or E. coli prevented pathogen-induced cell death at rates comparable to LGG. In contrast, L33 failed to exert a protective effect. Moreover, L125 significantly reduced adherence of S. aureus to HT-29 cells, and the internalization capacity of both pathogens (>1.5 Log CFU/mL). Dual RNA-seq and protein microarrays were used to determine expression changes in L125 and host cells during co-incubation. L125 expressed high levels of adhesins and moonlighting proteins, homologous to those encoded by the pathogens. Pathways involved in pathogen adhesion and internalization, endocytosis, cell–cell and cell-extracellular matrix (ECM) adhesion, were downregulated in HT-29 cells. Finally, L125 reduced the secretion of various pro-inflammatory mediators. Our findings highlight the strain-specific protective effects of LAB against pathogen-induced cell death achieved through competitive exclusion and priming of host cell responses. Future studies will focus on elucidating the specific surface components of L125 involved in these events, paving the way for targeted interventions at the host-pathogen interface.

2.3 Cell and bacteria viability assays

The cytotoxic capacity of pathogen CM was also evaluated using the HoloMonitor Live Imaging System (PHI, Boston, MA, United States). In this case, 2 × 105 cells per well were seeded into 24-well plates. The following day, cells were treated with undiluted, sterile-filtered pathogen CM. Cells treated with cell culture medium were included as an untreated control. Cell proliferation was assessed over a 24 h period with the Kinetic Cell Proliferation Assay software (PHI).

3.1 Staphylococcus aureus and Escherichia coli induce cell death of HT-29 cells in a time-dependent manner

These effects were further monitored for 24 h with the HoloMonitor live cell imaging system, using the Kinetic Cell Proliferation Assay software. It was shown that CM derived from either pathogen reduced cell confluency in a time dependent manner (Figure 1F).

(F) The effect of pathogen-derived CM was monitored for 24 h using the HoloMonitor live cell imaging system. SA CM and EC CM decreased confluency in HT-29 cells in a time-dependent manner. The data presented are the mean ± standard deviation of three independent experiments. *p < 0.05 and **p < 0.005 compared to control (CTRL) untreated cells.
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4. 22 kinesiska forskare har fått sina studier om elakartade cancertumörer har haft påverkan av ett specifikt kroppseget protein (SAMHD1) och om så hitta motmedel.

Nuclear Overexpression of SAMHD1 Induces M Phase Stalling in Hepatoma Cells and Suppresses HCC Progression by Interacting with the Cohesin Complex

Emerging evidence suggests that the sterile alpha-motif (SAM) and histidine aspartate (HD)domain-containingprotein1(SAMHD1)isimplicatedinvarious cancers, including hepatocellular carcinoma (HCC). However, its precise role in tumor cells and the underlying mechanisms remain unclear. This study aimed to investigate the expression patterns, prognostic values, and functional role of SAMHD1inHCCprogression. We constructed liver tissue microarrays using tumor and paired paratumor tissue specimens from 187 patients with primary HCC. Our findings indicate that nuclear SAMHD1 protein levels are increased in tumors compared to paratumor tissues. Moreover, nuclear SAMHD1 levels decline in advanced tumor stages, with higher SAMHD1 nuclear staining correlating with favorable prognostic outcomes. Hepatocyte-specific SAMHD1knockout mice, generated by crossing SAMHD1fl/fl mice with Alb-cre mice, showed accelerated tumor progression in a diethylnitrosamine (DEN)-induced HCC model. In hepatoma cell lines, nuclear overexpression of SAMHD1 inhibited cell proliferation by stalling mitosis, independent of its deoxynucleotide triphosphohydrolase (dNTPase) function. Mechanistically, SAMHD1 interacts with the cohesin complex in nucleus, enhancing sister chromatid cohesion during cell division, which delays metaphase progression. Our findings suggest that nuclear SAMHD1 plays a critical role in slowing HCC progression by regulating mitosis, highligh

Cell migration was evaluated using a wound healing assay and a Tran swell assay. For the wound healing assay, cells were seeded in 6-well plates and grown to confluence. A scratch was made with a sterile pipette tip, followed by washing to remove detached cells. Wound closure was mon itored over time using phase-contrast microscopy, and images were ana lyzed with ImageJ to calculate the percentage of wound closure relative to the initial area. For the Transwell assay, cells were resuspended in serum free DMEM at a density of 7.5 × 104 cells mL−1. A total of 200 μL of this suspension was added to the upper chamber, with 500 μL of DMEM con taining 20% FBS in the lower chamber. After 24 h of incubation, migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. 

To assess the impact of cohesion complex knockdown on the pro liferation and migration of SAMHD1-overexpressing HepG2 cells, cells were suspended in a medium containing a mixture of RNAiMax transfec tion reagent with scrambled siRNA or RAD21 and SMC3 siRNA mixture and were seeded in 6-well plates at 20% confluence for 24 h. Then the cell plates were placed into the HoloMonitor M4 digital holographic mi croscopy (Phase Holographic Imaging PHI Inc) within a standard CO2 in cubator. Six regions of interest (ROI) in each well were randomly selected by the instrument software, and images were captured every 2 h for a con tinuous 52-h period. 
The HoloMonitor software’s Kinetic Cell Proliferation Assay and Kinetic Cell Motility Assay modules were used to quantify cell growth and calculate average migration speed based on the captured im ages.

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5. 9 forskare från kända John Hopkins University USA har genomfört riktigt avancerade studier på celler och deras förmåga till anpassning? vid yttre påverkan. Forskning på allra högsta nivå känns adekvat för en lekman som jag att säga. Bedöm själva genom att läsa hela studien. Och av intresse: De pratar om QPM som teknik (Quantative Phase Microscopy) de använt för sina resultat. Med det menat att de använt HoloMonitor som vi kanske bättre känner igen som QPI. Närmare bestämt HoloMonitor tillsammans med Single Cell assay:en.

Cells Prioritize the Regulation of Cell Mass Density

I Introduction

How mammalian cells coordinate growth and maintain size across changing environments is a fundamental question in cell biology.
Recent advances in single-cell methods have enabled precise measurements of cell volume, mass, and cell mass density (CMD). For instance, microfluidic-based methods such as the Fluorescence eXclusion method (FXm) can accurately measure cell volume in the 10-100 femto-liter range.Micro-cantilever methods have been employed to measure cell dry mass in the femto-gram (fg) range.
Optical methods such as the Quantitative Phase Microscopy (QPM) can also accurately determine the dry mass (without water) of single cells with pico-gram sensitivity.Therefore, an unprecedented window is opening for examining live cell mass and volume, and also the cell mass density (mass/volume). In this paper, utilizing a combination of FXm and QPM, we quantitatively explore CMD regulation after media osmolarity change. We discover that while complex changes occur in cell volume and mass after osmotic shock, cells prioritize CMD recovery in a straightforward manner, suggesting that there is a simple control algorithm of CMD.

Quantitative phase microscopy

Cell dry mass represents the total mass of all intracellular non-water molecules. To measure cell dry mass, we utilized the Holomonitor M4 quantitative phase microscope (PHI). Quantitative phase microscopy (QPM) is based on the principle that the refractive index of a material inversely correlates with the velocity of light passing through it. When in operation, the Holomonitor directs a laser beam through both the cell and the surrounding culture medium. As light passes through the cell, which has a higher refractive index than the medium, it slows down, resulting in a detectable phase shift. This phase shift is captured by the camera and processed to summarize the total phase alteration caused by the entire cell (optical volume). The total phase alteration of individual cells was analyzed using the dedicated Holomonitor M4 software. For aqueous solutions, the refractive index is linearly proportional to the solute mass concentration, with a consistent factor α = 0.18 mL/g irrespective of the solute type.We have also validated this factor using a NaCl and a BSA solution. After obtaining the cell volume, the optical volume, and culture medium refractive index, cell mass can be calculated as:Embedded Imagewhere Δφ is the phase shift cells make in each pixel, nm is refractive index of cell culture medium, nwater is refractive index of water. The refractive index of different media was measured using the Digital Brix Refractometer (MILWAUKEE Instruments MA871).

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6. Som kronan på verket kommer slutligen en tvättäkta HoloMonitorbaserad studie där 7 polska forskare verkligen gått på djupet med genstudier (miR-1244) kopplat till cancer. Hänvisningarna till HoloMonitor är för många att ta upp så det blir ett urval. Studien är publicerad hos det förnämsta vetenskapliga organet av de alla : Springer Nature

The Yin and Yang of hsa-miR-1244 expression levels during activation of the UPR control cell fate

Abstract

Regulation of endoplasmic reticulum (ER) homeostasis plays a critical role in maintaining cell survival. When ER stress occurs, a network of three pathways called the unfolded protein response (UPR) is activated to reestablish homeostasis. While it is known that there is cross-talk between these pathways, how this complex network is regulated is not entirely clear. Using human cancer and non-cancer cell lines, two different genome-wide approaches, and two different ER stress models, we searched for miRNAs that were decreased during the UPR and surprisingly found only one, miR-1244, that was found under all these conditions. We also verified that ER-stress related downregulation of miR-1244 expression occurred with 5 different ER stressors and was confirmed in another human cell line (HeLa S3). These analyses demonstrated that the outcome of this reduction during ER stress supported both IRE1 signaling and elevated BIP expression. Further analysis using inhibitors specific for IRE1, ATF6, and PERK also revealed that this miRNA is impacted by all three pathways of the UPR. This is the first example of a complex mechanism by which this miRNA serves as a regulatory check point for all 3 pathways that is switched off during UPR activation. In summary, the results indicate that ER stress reduction of miR-1244 expression contributes to the pro-survival arm of UPR.

Real-time cell viability assay

For real-time monitoring of cell viability, we applied real-time and label-free holographic microscopy-based monitoring of cell death and viability using HoloMonitor M4® time-lapse cytometer (Phase Holographic Imaging PHI AB, Lund, Sweden). 
Holographic microscopy was used to follow the optical thickness and irregularity of cells exposed for up to 24 h to Tm or Tg in the presence or absence of miR-1244 mimic or antagomiR. The images from up to 8 independent optical fields were collected and analyzed according to manufactures instructions with HoloMonitor® App Suite software. Healthy cells are irregular in shape and thin, whereas dying cells are round and thick. For all analysis, the same cells parameters qualification was applied.
To better understand this the cell fate decision dynamics, we performed real time and label free holographic microscopy-based monitoring of cell death and viability using a HoloMonitor® time-lapse cytometer. Holographic microscopy was used to follow the optical thickness and irregularity of cells exposed for up to 24 h to Tm and Tg in the presence or absence of miR-1244 mimic or antagomiR (Figs. 45 and 6 and Supplemental Figs. 68).
Exogenous miR-1244 influences the fate of cells challenged with tg induced ER stress. The results of real-time monitoring of cell viability with the real time and label free holographic microscopy are shown using a HoloMonitor M4® time-lapse cytometer of HeLa cells transfected with miR-1244 mimic or inhibitor or the scramble control and 48 h later treated with Tg (500 nM) up to 24 h. Images were collected every 15 min (from 5 independent optical fields), and the distribution of live (blue) and dying cells (red) based on their optical thickness (Y-axis) and irregularity (X-axis) is presented at the 0, 8, 16 and 24 h time points. The images from up to 5 independent optical fields were collected and analyzed according to manufacturer’s instructions with HoloMonitor® App Suite software.

Genstudier med HoloMonitor !

Min kommentar
HoloMonitor avslutar detta år med 6 forskningsrapporter där 2 är extra vassa och förmodligen kommer användas i PHI`s marknadsföring. Ett år som för oss aktieägare inte varit nån höjdare ska ärligt sägas.
Men instrumentet skördar nya segrar som synes och det vore väl fasiken om inte vår nya ledning ska kunna kapitalisera på det konstaterandet. 2025 ska bli året då PHI och aktien gör en hejdundrande come back har jag bestämt. 😎

                                                 Mvh the99


Som service till alla HoloMonitornyfikna forskare : HoloMonitor Demo

onsdag 18 december 2024

Svensk Life Science - 22/1 2025

Göteborgsbaserade CCRM Nordic jag skrivit om här förärar Stockholm,Karolinska Institutet ett besök.
CCRM kommer den 22 Jan tillsammans med NextGenNK genomföra ett minisymposium om senaste utvecklingen inom ATMP (Advanced Therapy Medicinal Products). Det hos Sveriges senaste/nya Klustermotor Flemingsberg. Ett EU finansierat kluster som ska hjälpa till att sätta svensk Life Science på kartan.
📣 Vi är glada över att meddela att vi har beviljats EU-stöd för projektet Klustermotor Flemingsberg – en satsning för att stärka tillväxten inom life science!

Flemingsberg tar ett stort kliv mot att etablera sig som en central aktör inom Life Science med lanseringen av projektet ”Klustermotor Life Science Flemingsberg”. Projektet har beviljats medel från europeiska regionala utvecklingsfonden och syftar till att stärka tillväxten och etableringen av små och medelstora företag (SMF) inom life science-sektorn i Stockholmsregionen. Den totala budgeten på projektet är 10,8 miljoner för åren 2025-2027.

NextGenNK är navet för kommande svenska upptäckter baserade på avancerad cellforskning med mål att eliminera cancer mer effektivt än idag möjligt. 

Som de beskriver enligt :

Vision

Our vision is to transform tomorrow’s means of treating cancer by innovative and affordable NK cell-based therapies developed in collaboration with SME/industry and health care, and thereby, make Swedish and select international SMEs/industry global leaders in the field of cancer immunotherapy.
The presently outlined therapies will potentially transform medical practice, with the promise to treat, manage and potentially cure some of the most debilitating and costly diseases. As such, we aim to create and develop a new segment in Swedish Life Science Industry Research Strategy
NextGenNK and CCRM Nordic mini-symposium
Advanced Medicinal Therapy Products, ATMP, span broadly over medical fields and the aim of this symposium is to enhance networking within ATMP and to get a flavor of academic and commercial development of ATMPs in Stockholm and Sweden. NextGenNK and CCRM Nordic are inviting you to a mini-symposium with the focus on ATMP on January 22, 2025. 

ATMP Sweden som PHI är medlem i skriver såhär om detta mini-symposium :
🔬✨ Strengthening Sweden's ATMP Ecosystem! ✨🔬

We’re excited to see member organizations like CCRM Nordic, in collaboration with NextGenNK, driving important initiatives like the ATMP Mini-Symposium on January 22, 2025, at Karolinska Institutet’s ATMP Flemingsberg Campus.

This symposium will provide a valuable platform for exchanging insights and building connections within the Swedish ATMP community, highlighting the exciting academic and commercial developments in Stockholm and beyond.

Hope to see you there! 💫


Min kommentar
Svenska Regeringens initiativ att satsa på Svensk Life Science har dragit igång. Månne PHI kan hitta en bra corner i detta? Som leverantör av Svensk högteknologisk teknik ypperligt passande Svensk avancerad cellforskning. Mvh the99