DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

Mitocurcumin utilizes oxidative stress to upregulate JNK/p38 signaling and overcomes Cytarabine resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer that is characterized by the rapid growth of abnormal myeloid cells. The goal of AML treatment is to eliminate the leukemic blasts, which is accomplished through intensive chemotherapy. Cytarabine is a key component of the standard induction chemotherapy regimen for AML. However, despite a high remission rate, 70-80% of AML patients relapse and develop resistance to Cytarabine, leading to poor clinical outcomes. Mitocurcumin (MitoC), a derivative of curcumin that enters mitochondria, leading to a drop in mitochondrial membrane potential and mitophagy induction. Further, it activates oxidative stress-mediated JNK/p38 signaling to induce apoptosis. MitoC demonstrated a preferential ability to kill leukemic cells from AML cell lines and patient-derived leukemic blasts. RNA sequencing data suggests perturbation of DNA damage response and cell proliferation pathways in MitoC-treated AML. Elevated reactive oxygen species (ROS) in MitoC-treated AML cells resulted in significant DNA damage and cell cycle arrest. Further, MitoC treatment resulted in ROS-mediated enhanced levels of p21, which leads to suppression of CHK1, RAD51, Cyclin-D and c-Myc oncoproteins, potentially contributing to Cytarabine resistance. Combinatorial treatment of MitoC and Cytarabine has shown synergism, increased apoptosis, and enhanced DNA damage. Using AML xenografts, a significant reduction of hCD45+ cells was observed in AML mice bone marrow treated with MitoC (mean 0.6%; range0.04%-3.56%) compared to control (mean 38.2%; range10.1%-78%), p = 0.03. The data suggest that MitoC exploits stress-induced leukemic oxidative environment to up-regulate JNK/p38 signaling to lead to apoptosis and can potentially overcome Cytarabine resistance via ROS/p21/CHK1 axis.

Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax.

INTRODUCTION: The emergence of resistance to the highly successful BCL2-directed therapy is a major unmet need in acute myeloid leukemia (AML), an aggressive malignancy with poor survival rates. Towards identifying therapeutic options for AML patients who progress on BCL2-directed therapy, we studied a clinical-stage CDK7 inhibitor XL102, which is being evaluated in solid tumors (NCT04726332). MATERIALS AND METHODS: To determine the anti-proliferative effects of XL102, we performed experiments including time-resolved fluorescence resonance energy transfer, target occupancy, cell cycle and apoptosis-based assays. We also included genetically characterized primary myeloid blasts from de novo and relapsed/refractory AML patients. For mechanistic studies, CRISPR/Cas9 mediated knockout of CDK7 and c-Myc and immunoblotting were performed. NOD/SCID orthotropic and subcutaneous AML xenografts were used to determine anti-leukemic effects. To assess the synergistic effects of XL102 with Venetoclax, we performed RNA sequencing and gene set enrichment analysis using Venetoclax sensitive and resistant model systems. RESULTS: XL102, a highly specific, orally bioavailable covalent inhibitor of CDK7. Inhibitory effect on CDK7 by XL102 in primary myeloid blasts (n = 54) was in nanomolar range (mean = 300 nM; range = 4.0-952 nM). XL102 treated AML cells showed a reduction in phosphorylation levels of Serine 2/5/7 at carboxy-terminal domain of RNA polymerase II. T-loop phosphorylation of CDK1(Thr161) and CDK2(Thr160) was inhibited by XL102 in dose-dependent manner leading to cell-cycle arrest. c-Myc downregulation and enhanced levels of p53 and p21 in XL102 treated cells were observed. Increased levels of p21 and activation of p53 by XL102 were mimicked by genetic ablation of CDK7, which supports that the observed effects of XL102 are due to CDK7 inhibition. XL102 treated AML xenografts showed remarkable reduction in hCD45 + marrow cells (mean = 0.60%; range = 0.04%-3.53%) compared to vehicle control (mean = 38.2%; range = 10.1%-78%), with corresponding increase in p53, p21 and decrease in c-Myc levels. The data suggests XL102 induces apoptosis in AML cells via CDK7/c-Myc/p53 axis. RNA-sequencing from paired Venetoclax-sensitive and Venetoclax-resistant cells treated with XL102 showed downregulation of genes involved in proliferation and apoptosis. CONCLUSION: Taken together, XL102 with Venetoclax led to synergistic effects in overcoming resistance and provided a strong rationale for clinical evaluation of XL102 as a single agent and in combination with Venetoclax.

KDEON WK-11: A short antipseudomonal peptide with promising potential.

The plight of antimicrobial resistance continues to limit the availability of antibiotic treatment effective in combating resistant bacterial infections. Despite efforts made to rectify this issue and minimise its effects on both patients and the wider community, progress in this area remains minimal. Here, we de-novo designed a peptide named KDEON WK-11, building on previous work establishing effective residues and structures active in distinguished antimicrobial peptides such as lactoferrin. We assessed its antimicrobial activity against an array of bacterial strains and identified its most potent effect, against Pseudomonas aeruginosa with an MIC value of 3.12 µM, lower than its counterparts developed with similar residues and chain lengths. We then determined its anti-biofilm properties, potential mechanism of action and in vitro cytotoxicity. We identified that KDEON WK-11 had a broad range of antimicrobial activity and specific capabilities to fight Pseudomonas aeruginosa with low in vitro cytotoxicity and promising potential to express anti-lipopolysaccharide qualities, which could be exploited to expand its properties into an anti-sepsis agent.

Genetic ablation of pregnancy zone protein promotes breast cancer progression by activating TGF-ß/SMAD signaling.

PURPOSE: Pregnancy zone protein (PZP) is best known as protease inhibitor and its concentration in human blood plasma increases dramatically during pregnancy. Recent investigation revealed a role of PZP inactivating germ-line mutation in breast cancer predisposition, and therefore we designed a study to evaluate functional involvement of this protein in tumor pathogenesis. METHODS: PZP knockout cells were generated utilizing the CRISPR-Cas9 approach in MCF7 and T47D (breast cancer) cell lines, and colony formation, cell proliferation, and migration assays carried out. TGF-ß and SMAD expression studies were performed using qRT-PCR and Western blot. PZP expression in tumor vs normal tissue was compared using meta-analyses of data records of breast cancer patients (n = 1211) included in the TCGA consortium registry as well as in independent cohorts of hormone receptor-positive (n = 118) and triple-negative breast cancer (TNBC) patients (n = 116). RESULTS: We demonstrated that genetic ablation of PZP efficiently inhibits tamoxifen-induced apoptosis and enhances cell proliferation, migration, and colony-forming capacity. We found a significant increase in survival fraction of CRISPR/Cas9-mediated PZP knockout clones compared to wild-type counterpart after tamoxifen treatment (p < 0.05). The PZP knockout significantly promoted breast cancer cell migration (p < 0.01) in vitro. We observed high expression of TGF-ß2 ligand, TGF-ß- receptor 2, and upregulation of phosphorylated regulatory-SMADs (pSMAD2 and pSMAD3) activating the pro-survival function of TGF-ß/SMAD signaling in PZP knockout clones. Meta-analyses of data records of breast cancer patients indicated that low PZP expression is associated with poor overall survival at 6 years (51.7% vs 62.9% in low vs high expressers, respectively; p = 0.026). We also observed a significantly lower PZP mRNA expression in TNBC as compared with hormone receptor-positive tumors (p = 0.019). CONCLUSION: Taken together, our results suggest that genetic ablation of PZP results in tumor progression and low expression of PZP is associated with poor survival of breast cancer patients.

Characterization of therapy-related acute leukemia in hereditary breast-ovarian carcinoma patients: role of BRCA1 mutation and topoisomerase II-directed therapy.

Therapy-related acute leukemias (t-ALs) represent approximately 10-20% of all acute leukemias, are frequently resistant to chemotherapy, and are associated with guarded outcomes. The national comprehensive cancer network data suggest that t-AL cases are diagnosed at increasing rates in breast cancer patients treated with chemotherapeutic agents targeting topoisomerase II. Two cases of BRCA1-mutated ovarian and breast carcinoma who developed therapy-related APL and ALL, respectively, following topoisomerase II-directed therapy were characterized. Genomic characterization of therapy-related acute promyelocytic leukemia (t-APL) revealed a unique RARA intron 2 breakpoint (Chr17: 40347487) at 3'-end of RARA corroborating breakpoint clustering in t-APL following topoisomerase II inhibition. Both cases of this series harbored germline BRCA1 mutations. The germline BRCA1 mutation in patient with t-APL was detected in exon 8 (HGVS nucleotide: c.512dupT). This mutation in t-APL is extremely rare. Interestingly, t-ALL patient in this series had a BRCA1 mutation (HGVS nucleotide: c.68_69delAG; BIC designation: 187delAG) identical to a previously reported case after the treatment of same primary disease. It is unlikely that two breast cancer patients with identical BRCA1 mutation receiving topoisomerase II-targeted agents for the primary disease developed t-AL by chance. This report highlights the development of t-AL in BRAC1-mutated hereditary breast and ovarian cancer patients and warrants further studies on functional consequences of topoisomerase inhibition in this setting.

miRNA-mRNA Profiling Reveals Prognostic Impact of SMC1A Expression in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) with NPM1 mutation is a disease driving genetic alteration with good prognosis. Although it has been suggested that NPM1 mutation induces chemosensitivity in leukemic cells, the underlying cause for the better survival of NPM1 mutated patients is still not clear. Mutant NPM1 AML has a unique microRNA and their target gene (mRNA) signature compared to wild-type NPM1. Dynamic regulation of miRNA-mRNA has been reported to influence the prognostic outcome. In the present study, in silico expression data of miRNA and mRNA in AML patients was retrieved from genome data commons, and differentially expressed miRNA and mRNA among NPM1 mutated (n = 21) and NPM1 wild-type (n = 162) cases were identified to establish a dynamic association at the molecular level. In vitro experiments using high-throughput RNA sequencing were performed on human AML cells carrying NPM1 mutated and wild-type allele. The comparison of in vitro transcriptomics data with in silico miRNA-mRNA expression network data revealed downregulation of SMC1A. On establishing miRNA-mRNA interactive pairs, it has been observed that hsa-mir-215-5p (logFC: 0.957; p = 0.0189) is involved in the downregulation of SMC1A (logFC: -0.481; p = 0.0464) in NPM1 mutated AML. We demonstrated that transient expression of NPM1 mutation upregulates miR-215-5p, which results in downregulation of SMC1A. We have also shown using a rescue experiment that neutralizing miR-215-5p reverses the effect of NPM1 mutation on SMC1A. Using the leukemic blasts from AML patients, we observed higher expression of miR-215-5p and lower expression of SMC1A in NPM1 mutated patients compared to wild-type cases. The overall survival of AML patients was significantly inferior in SMC1A high expressers compared to low expressers (20.3% vs. 58.5%, p = 0.018). The data suggest that dynamic miR-215-SMC1A regulation is potentially modulated by NPM1 mutation, which might serve as an explanation for the better outcome in NPM1 mutated AML.

Exome sequencing study of Russian breast cancer patients suggests a predisposing role for USP39.

PURPOSE: Germline variants in known breast cancer (BC) predisposing genes explain less than half of hereditary BC cases. This study aimed to identify missing genetic determinants of BC. METHODS: Whole exome sequencing (WES) of lymphocyte DNA was performed for 49 Russian patients with clinical signs of genetic BC predisposition, who lacked Slavic founder mutations in BRCA1, BRCA2, CHEK2, and NBS1 genes. RESULTS: Bioinformatic analysis of WES data was allowed to compile a list of 229 candidate mutations. 79 of these mutations were subjected to a three-stage case-control analysis. The initial two stages, which involved up to 797 high-risk BC patients, 1504 consecutive BC cases, and 1081 healthy women, indicated a potentially BC-predisposing role for 6 candidates, i.e., USP39 c.*208G > C, PZP p.Arg680Ter, LEPREL1 p.Pro636Ser, SLIT3 p.Arg154Cys, CREB3 p.Lys157Glu, and ING1 p.Pro319Leu. USP39 c.*208G > C was strongly associated with triple-negative breast tumors (p = 0.0001). In the third replication stage, we genotyped the truncating variant of PZP (rs145240281) and the potential splice variant of USP39 (rs112653307) in three independent cohorts of Russian, Byelorussian, and German ancestry, comprising a total of 3216 cases and 2525 controls. The data obtained for USP39 rs112653307 supported the association identified in the initial stages (the combined OR 1.72, p = 0.035). CONCLUSIONS: This study suggests the role of a rare splicing variant in BC susceptibility. USP39 encodes an ubiquitin-specific peptidase that regulates cancer-relevant tumor suppressors including CHEK2. Further epidemiological and functional studies involving these gene variants are warranted.

Molecular Heterogeneity in Acute Promyelocytic Leukemia - a Single Center Experience from India.

Atypical breakpoints and variant APL cases involving alternative chromosomal aberrations are seen in a small subset of acute promyelocytic leukemia (APL) patients. Over seven different partner genes for RARA have been described. Although rare, these variants prove to be a diagnostic challenge and require a combination of advanced cytogenetic and molecular techniques for accurate characterization. Heterogeneity occurs not only at the molecular level but also at clinico-pathological level influencing treatment response and outcome. In this case series, we describe the molecular heterogeneity of APL with a focus on seven variant APL cases from a single tertiary cancer center in India over a period of two and a half years. We discuss five cases with ZBTB16-RARA fusion and two novel PML-RARA variants, including a Bcr3 variant involving fusion of PML exon4 and RARA exon3 with an additional 40 nucleotides originating from RARA intron2, another involving exon 6 of PML and exon 3 of RARA with addition of 126 nucleotides, which mapped to the central portion of RARA intron 2. To the best of our knowledge, this is the first case series of this kind from India.

Inhibition of novel GCN5-ATM axis restricts the onset of acquired drug resistance in leukemia.

Leukemia is majorly treated by topoisomerase inhibitors that induce DNA double strand breaks (DSB) resulting in cell death. Consequently, modulation of DSB repair pathway renders leukemic cells resistant to therapy. As we do not fully understand the regulation of DSB repair acquired by resistant cells, targeting these cells has been a challenge. Here we investigated the regulation of DSB repair pathway in early drug resistant population (EDRP) and late drug resistant population (LDRP). We found that doxorubicin induced equal DSBs in parent and EDRP cells; however, cell death is induced only in the parent cells. Further analysis revealed that EDRP cells acquire relaxed chromatin via upregulation of lysine acetyl transferase KAT2A (GCN5). Drug treatment induces GCN5 interaction with ATM facilitating its recruitment to DSB sites. Hyperactivated ATM maximize H2AX, NBS1, BRCA1, Chk2, and Mcl-1 activation, accelerating DNA repair and survival of EDRP cells. Consequently, inhibition of GCN5 significantly reduces ATM activation and survival of EDRP cells. Contrary to EDRP, doxorubicin failed to induce DSBs in LDRP because of reduced drug uptake and downregulation of TOP2ß. Accordingly, ATM inhibition prior to doxorubicin treatment completely eliminated EDRP but not LDRP. Furthermore, baseline AML samples (n = 44) showed significantly higher GCN5 at mRNA and protein levels in MRD positive compared to MRD negative samples. Additionally, meta-analysis (n = 221) showed high GCN5 expression correlates with poor overall survival. Together, these results provide important insights into the molecular mechanism specific to EDRP and will have implications for the development of novel therapeutics for AML.

Studies of protein-protein interactions in Fanconi anemia pathway to unravel the DNA interstrand crosslink repair mechanism.

Fanconi anemia (FA), a cancer predisposition syndrome exhibits hallmark feature of radial chromosome formation, and hypersensitivity to DNA crosslinking agents. A set of FA pathway proteins mainly FANCI, FANCD2 and BRCA2 are expressed to repair the covalent crosslink between the dsDNA. However, FA, BRCA pathways play an important role in DNA ICL repair as well as in homologous recombination repair, but the presumptive role of FA-BRCA proteins has not clearly explored particularly in context to function associated protein-protein interactions (PPIs). Here, in-vivo, in-vitro and in-silico studies have been performed for functionally relevant domains of FANCI, FANCD2 and BRCA2. To our conclusion, FANCI ARM repeat interacts with FANCD2 CUE domain and BRCA2 C-terminal region. Interestingly, FANCD2 CUE domain also interacts strongly with BRCA2 C-terminal region. Interactions between BRCA2 CTR and functionally relevant mutations Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant) present in FANCD2 CUE domain have been analysed. To our finding, these mutations abrogate the binding between FANCD2 CUE domain and BRCA2 CTR. Furthermore, (1) different domain of FANCI, FANCD2 and BRCA2 are playing important role in PPIs, (2) mutations cause the impairment in the PPIs which in turn may disrupt the DNA ICL repair mechanism.

Structural and biophysical properties of h-FANCI ARM repeat protein.

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

The hidden genomic landscape of acute myeloid leukemia: subclonal structure revealed by undetected mutations.

The analyses carried out using 2 different bioinformatics pipelines (SomaticSniper and MuTect) on the same set of genomic data from 133 acute myeloid leukemia (AML) patients, sequenced inside the Cancer Genome Atlas project, gave discrepant results. We subsequently tested these 2 variant-calling pipelines on 20 leukemia samples from our series (19 primary AMLs and 1 secondary AML). By validating many of the predicted somatic variants (variant allele frequencies ranging from 100% to 5%), we observed significantly different calling efficiencies. In particular, despite relatively high specificity, sensitivity was poor in both pipelines resulting in a high rate of false negatives. Our findings raise the possibility that landscapes of AML genomes might be more complex than previously reported and characterized by the presence of hundreds of genes mutated at low variant allele frequency, suggesting that the application of genome sequencing to the clinic requires a careful and critical evaluation. We think that improvements in technology and workflow standardization, through the generation of clear experimental and bioinformatics guidelines, are fundamental to translate the use of next-generation sequencing from research to the clinic and to transform genomic information into better diagnosis and outcomes for the patient.

Biology and management of therapy-related acute promyelocytic leukemia.

PURPOSE OF REVIEW: Therapy-related acute promyelocytic leukemia (t-APL) has been increasingly reported after exposure to cytotoxic and/or immunosuppressive agents given for prior malignancies or autoimmune diseases. t-APL represents both a model for better understanding human leukemogenesis and an interesting therapeutic subset which requires specific adaptations for optimal management. RECENT FINDINGS: We discuss here potential risk factors for t-APL development and the main biologic and clinical characteristics of t-APL as compared to de-novo APL.In addition, we review therapeutic results obtained in patients with t-APL receiving conventional retinoic acid and chemotherapy and discuss new treatment opportunities with minimal or no exposure to conventional cytotoxic agents. SUMMARY: Genomic studies in patients at risk of t-APL are relevant to better adapt treatment for the primary disease and to implement monitoring during follow-up and early diagnosis of t-APL. Improved molecular characterization of t-APL may include next generation sequencing approaches to better identify distinguishing features as compared to de-novo APL. Early diagnosis of t-APL through careful monitoring of patients at higher risk, coupled to incorporation in the therapeutic armamentarium of novel effective agents such as arsenic trioxide could result in improved clinical outcome for these patients.

Evidence for direct involvement of epirubicin in the formation of chromosomal translocations in t(15;17) therapy-related acute promyelocytic leukemia.

Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17)(q22;q21) involving the PML and RARA genes is associated with exposure to agents targeting topoisomerase II (topoII), particularly mitoxantrone and epirubicin. We previously have shown that mitoxantrone preferentially induces topoII-mediated DNA damage in a "hotspot region" within PML intron 6. To investigate mechanisms underlying epirubicin-associated t-APL, t(15;17) genomic breakpoints were characterized in 6 cases with prior breast cancer. Significant breakpoint clustering was observed in PML and RARA loci (P = .009 and P = .017, respectively), with PML breakpoints lying outside the mitoxantrone-associated hotspot region. Recurrent breakpoints identified in the PML and RARA loci in epirubicin-related t-APL were shown to be preferential sites of topoII-induced DNA damage, enhanced by epirubicin. Although site preferences for DNA damage differed between mitoxantrone and epirubicin, the observation that particular regions of the PML and RARA loci are susceptible to these agents may underlie their respective propensities to induce t-APL.

Layer by layer three-dimensional tissue epitaxy by cell-laden hydrogel droplets.

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm x 5 mm x 81 microm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 microm thick per layer) with controlled spatial resolution (proximal axis: 18.0 +/- 7.0 microm and distal axis: 0.5 +/- 4.9 microm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 +/- 2 cells/mm(2) at 1 million cells/mL, 122 +/- 20 cells/mm(2) at 5 million cells/mL, and 216 +/- 38 cells/mm(2) at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues.

Microfluidics for cryopreservation.

Minimizing cell damage throughout the cryopreservation process is critical to enhance the overall outcome. Osmotic shock sustained during the loading and unloading of cryoprotectants (CPAs) is a major source of cell damage during the cryopreservation process. We introduce a microfluidic approach to minimize osmotic shock to cells during cryopreservation. This approach allows us to control the loading and unloading of CPAs in microfluidic channels using diffusion and laminar flow. We provide a theoretical explanation of how the microfluidic approach minimizes osmotic shock in comparison to conventional cryopreservation protocols via cell membrane transport modeling. Finally, we show that biological experiments are consistent with the proposed mathematical model. The results indicate that our novel microfluidic-based approach improves post-thaw cell survivability by up to 25% on average over conventional cryopreservation protocols. The method developed in this study provides a platform to cryopreserve cells with higher viability, functionality, and minimal inter-technician variability. This method introduces microfluidic technologies to the field of biopreservation, opening the door to future advancements at the interface of these fields.

AML1-ETO positive AML: first report from India.

Translocation (8;21) is associated with few typical morphological features and favorable prognosis. All patients of AML and MDS with increased blasts (N = 35) according to FAB criteria, presenting (between Jan 2004 to June 2005) to the Department of Hematology, AIIMS were studied. RT-PCR was done for the AML1-ETO fusion transcript in all cases. Overall incidence of AML1-ETO was 28.57% and no correlation was found between AML1-ETO positivity and clinical or hematological parameters except for a direct correlation with absolute blast count (ABC) (a lower ABC in the AML1-ETO positive cases). Interestingly, 1/3 MDS cases were positive for the same fusion transcript and thus, it appears worthwhile to look for AML1-ETO in all cases of MDS with increased blasts. Objective morphological evaluation using a scoring system based on morphological features was not helpful in predicting positivity for AML1-ETO. The effect of this translocation on long-term survival could not be determined by the present study.