Machine learning for enumeration of cell colony forming units

As one of the most widely used assays in biological research,an enumeration of the bacterial cell colonies is an important but time-consuming and labor-intensive process.To speed up the colony counting,a machine learning method is presented for counting the colony forming units(CFUs),which is referred to as CFUCounter.This cellcounting program processes digital images and segments bacterial colonies.The algorithm combines unsupervised machine learning,iterative adaptive thresholding,and local-minima-based watershed segmentation to enable an accurate and robust cell counting.Compared to a manual counting method,CFUCounter supports color-based CFU classification,allows plates containing heterologous colonies to be counted individually,and demonstrates overall performance(slope 0.996,SD 0.013,95%CI:0.97–1.02,p value<1e-11,r=0.999)indistinguishable from the gold standard of point-and-click counting.This CFUCounter application is open-source and easy to use as a unique addition to the arsenal of colony-counting tools.

Direct Infection of Colony Forming Unit-Megakaryocyte by Human Cytomegalovirus Contributes the Pathogenesis of Idiopathic Thrombocytopenic Purpura

Human cytomegalovirus （HCMV） late mRNA expression in megakaryoblast and in turn the pathogenesis of idiopathic thrombocytopenic purpura （ITP） patients with HCMV infection, and effectiveness of ganciclovir were investigated. Colony forming unit-megakaryocytes （CFU-MK） of 46 ITP patients with HCMV infection were incubated from patients＇ bone marrow mononuclear cells （MNC）. Reverse transcriptase-polymerase chain reaction （RT-PCR） was subsequently used for CFU-MK for HCMV-late mRNA detection, Ganciclovir therapy was given to both HCMV-late mRNA positive and negative groups for comparison of therapeutic effectiveness, The results in 19 of 46 CFU-MK culture cells specimens with positive HCMV-DNA by PCR or positive CMV-IgM by enzyme linked immunosorbent assay （ELISA） in the correspondent serum of peripheral blood were positive for HCMV-late mRNA, Sixteen out of 19 patients with positive HCMV-late mRNA CFU-MK had a positive response to ganciclovir. Amongst 27 patients with negative HCMV-late mRNA CFU-MK, only 4 positive responders to ganciclovir therapy were observed. Curative effectiveness of ganciclovir in HCMV-late mRNA positive group was significantly higher than that in HCMV-late mRNA negative group （P〈0.01）, It was suggested that HCMV could directly infect CFU-MK, which might be one of the mechanisms responsible for HCMV related ITE The ganci- clovir is an effective therapy in resulting in the increases in thrombocyte in the ITP patients whose HCMV- late mRNA was positive in their CFU-MK.

Clonal isolation of endothelial colony-forming cells from early gestation chorionic villi of human placenta for fetal tissue regeneration

BACKGROUND Endothelial colony-forming cells(ECFCs)have been implicated in the process of vascularization,which includes vasculogenesis and angiogenesis.Vasculogenesis is a de novo formation of blood vessels,and is an essential physiological process that occurs during embryonic development and tissue regeneration.Angiogenesis is the growth of new capillaries from pre-existing blood vessels,which is observed both prenatally and postnatally.The placenta is an organ composed of a variety of fetal-derived cells,including ECFCs,and therefore has significant potential as a source of fetal ECFCs for tissue engineering.AIM To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi(CV-ECFCs)of the placenta,and assess their potential for tissue engineering.METHODS The early gestation chorionic villus tissue was dissociated by enzyme digestion.Cells expressing CD31 were selected using magnetic-activated cell sorting,and plated in endothelial-specific growth medium.After 2-3 wks in culture,colonies displaying cobblestone-like morphology were manually picked using cloning cylinders.We characterized CV-ECFCs by flow cytometry,immunophenotyping,tube formation assay,and Dil-Ac-LDL uptake assay.Viral transduction of CVECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector,and transduction efficiency was tested by fluorescent microscopy and flow cytometry.Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved,small intestinal submucosa extracellular matrix scaffold.RESULTS After four passages in 6-8 wks of culture,we obtained a total number of 1.8×107 CV-ECFCs using 100 mg of early gestational chorionic villus tissue.Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31,CD144,CD146,CD105,CD309,only partially expressed CD34,and did not express CD45 and CD90.CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation,similar to cord blood-derived ECFCs(CB-ECFCs).CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%.Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold.CONCLUSION In summary,we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs.A substantial number of CV-ECFCs can be obtained within a short time frame,representing a promising novel source of ECFCs for fetal treatments.

Microbiological Quality of Freshly Prepared, Packaged Fruit and Milk Juices Sold in Cafés, Shops, and Supermarkets in Hargeisa, Somaliland

Background: Due to their delicious taste, high nutritional content, and health benefits, fruit juices are well-known drinks in many countries and are now an essential component of the modern diet. Objective: Determining the microbiological quality of both packaged and freshly made fruit and milk juices. Method: The spread-plate approach was employed to isolate and count the bacteria. 90 ml of sterile peptone water were blended with 10 ml of well-mixed, packed, and freshly made fruit juices. The samples were sequentially diluted (101 - 105) in accordance with the Indian Manual of Food Microbiological Testing Methods. Results: From eight samples of imported packaged fruit and milk juice, the average of total coliform, staphylococci, and viable bacterial counts were zero, 1.39 × 102, and 2 × 102 CFU/ml, respectively. In contrast, from three samples of locally produced fruit and milk juice, the average of total coliform, staphylococci, and viable bacterial counts were zero, 5.83 × 102, and 2.73 × 103 CFU/ml, respectively. Four samples of handmade prepared fruit and milk juices had a mean of total coliform, staphylococci, and viable bacterial count of 1.441 × 104, 4.1 × 103, and 2.35 × 105 CFU/ml, respectively. Conclusion: 33.3% of the results from microbiological analysis of freshly made fruit and milk juices met the permissible range of the Revised Microbiological Standards for Fruit and Vegetables and Their Products, which were published in 2018 and as well as the Hong Kong Center for Food Safety, whereas 66.7% of the microbiological analyses of freshly prepared fruit and milk juices were above the permissible reference range of GSO standard 2000. 12.5% of the investigated imported and packed fruits and milk juices had one failed test (TSC), which was above the acceptable limit, 87.5% of the tested samples of fruit and milk juices fulfilled the necessary standards of TCC, TVBC, and TSC. 100% of the tested locally manufactured fruit and milk juices complied with TSC, TCC, and TVBC requirements. All investigations showed that freshly made fruit and milk juices were heavily contaminated (Total viable bacterial count, total coliform count, and total staphylococcus count). .

Study on the Effects of Guiqi Oral Liquid (归芪口服液) in Promoting Recovery of Hematopoiesis in AcuteIrradiation Injured Mice

Objective： To explore the effects and possible mechanisms of Guiqi Oral Liquid （归芪口服液, GQOL） on the recovery of hematopoiesis in acute irradiation injured mice. Methods： The acute irradiation injured mice were randomly divided into 2 groups： the treated group and the control group, and also a normal control group was set up with 6 mice in it receiving no treatment. After the mice in the former two groups were irradiated by 6.0 Gy ^60Coγ-ray, every one of them was given 0.4 ml GQOL or saline in equal volume through a gastric tube twice a day for 14 days. On the 4th, 8th and 14th day after irradiation, the bone marrow mononuclear cells （BMMNC） and megakaryocytes in bone marrow tissues of the mice were counted, the proportion of hematopoietic tissues （by area） was measured, and the expression of adhesion molecules, CD44 and CD54, in bone marrow were estimated by immunochemistry. The colony forming unit of spleen （CFU-S） in the mice were counted on the 8th day after irradiation. Results： On the 4th, 8th, 14th day after irradiation, the count of BMMNC and megakaryocyte, and the proportion of hematopoietic tissues in the treated group were higher than those in the control group （P〈0.01 or P〈0.05）. CD44 and CD54 expression in the treated group were higher than those in the control group on the 4th and 8th day （P〈0.01）, but near normal on the 14th day （P〈0.01）. On the 8th day, CFU-S count in the treated group was higher than that in the control group （P〈0.01）. Conclusion： GQOL can regulate the expression of adhesion molecules, CD44 and CD54, in the bone marrow of the acute irradiation injured mice, which may be one of the mechanisms of GQOL in accelerating the early phase hematopoiesis recovery of mice.

Effects of preservation time on proliferative potential of human limbal stem/progenitor cells

AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell markers. METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4 degrees C for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1st day and disappeared at the 4th day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days' preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.

Inhibition of Proliferation of Human Hela Cells by Small Interference RNA against Pokemon Gene

Objective： The transcriptional repressor Pokemon （encoded by the Zbtb7 gene） is a critical factor in oncogenesis. Pokemon overexpression leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice, The objective of this study was to investigate the effect of retrovirus expressing the siRNA targeting Pokemon in human cervical cancer cells. Methods： We constructed and identified the recombinant retrovirus particle expressing siRNA of Pokemon gene, and then testified the suppression of recombinant plasmid and evaluated the gene-silencing effect. Results： We got the positive evaluation from colony forming experiment we found that the retrovirus expressing siRNA targeting Pokemon had repressing effect. Conclusion： Our work provides basis for the study of suppression effect of retrovirus in vivo and the design of the target-complex.

Observation on Culture Characteristics of Mycoplasma bovis with Four Different Antigen Quantification Methods

[Objective] The paper was to explore the antigen harvest time of Mycoplasrna bovis and the antigen quantification alternative method. [Method] M. bovis 08M strain was inoculated in the Thiaueourt＇s medium containing 10% horse serum. Four growth curves were plotted by simultaneously measuring color change units （CCU）, colony forming units （CFU）, protein concentration and nucleic acid levels within 110 h. [ Result] The growth of M. bovis was divided into four phases： the longarithmie phase appeared after being cultured for 10 h; the stationary phase appeared at 30 h with the highest number of viable cells up to 1. 0× 108 CCU/mL and 7.7 × 107 CCU/mL, respectively; and the decline phase started at 75 h. The protein concentration afM. bov/s increased rapidly from 15 to 35 h, reached 72.06 μg/mL at 35 h, then maintained at 53.38 - 70.65 μg/mL. The nucleic acid levels of M. bov/s increased rapidly from 15 h, and the cycle threshold （Ct） values were maintained between 15, 32 and 17.84 after 25 h. [ Conclusion] There was a good correlation between the protein concentration and variable count of M. bov/s at the early stationary phase, which was the best time period to harvest antigen. The protein concentration determination could be an alternative method to quantify antigen content of M. bovis when preparing inactivated M. boviz vaccine. Key words Mycoplasma boyis; Antigen quantification; Color change units; Colony forming units; Protein concentration; Nucleic acid content

Microbial Analysis of Drinking Water from Randomly Selected Boreholes and Shallow Wells around Hargeisa, Somaliland

Background: Shallow wells and boreholes are vital sources of potable water in Hargeisa. This water can be polluted by runoff, in particular during the rainy season, causing outbreaks of waterborne infections. Objectives: This research aimed at evaluating the microbial quality of shallow wells and boreholes water around Hargeisa, Somaliland. Methods: The total coliform and Escherichia coli count were done by using the membrane filtration method. Overall, 100 ml of each water sample was filtered via a 0.45 μm membrane filter, and then the filters were put on m-Endo agar plates that were incubated at 37°C for 24 to 48 hours. Results: The mean value of total coliform counts for the boreholes and shallow wells ranged from 1.288 × 103 to 8.8 × 103 CFU/100ml, while the mean value of total E. coli counts also ranged from 3.5 × 102 to 4.429 × 103 CFU/100ml. Results from this study have demonstrated that all water sources (Arabsiyo, Dararweyne, Darasalaam, Dabaraqas, and Jaleelo) don’t comply with the WHO guideline for drinking water. Results from the analysis of water samples of 28 wells demonstrated a significant correlation between total coliform and E. coli counts (P = 0.01). Therefore, this water is not fit for human consumption unless it is treated. Conclusion: This study has demonstrated that all results of both mean values of total coliform and E. coli counts from groundwater of selected shallow wells and boreholes were beyond WHO standards, so water from Arabsiyo, Jaleelo, Dabaraqas, Dararweyne, and Darasalaam requires treatment before human consumption.

Desulfurization of dibenzothiophene(DBT) by a novel strain Lysinibacillus sphaericus DMT-7 isolated from diesel contaminated soil

A new bacterial strain DMT-7 capable of selectively desulfurizing dibenzothiophene（DBT） was isolated from diesel contaminated soil.The DMT-7 was characterized and identified as Lysinibacillus sphaericus DMT-7（NCBI GenBank Accession No.GQ496620） using 16S rDNA gene sequence analysis.The desulfurized product of DBT,2-hydroxybiphenyl（2HBP）,was identified and confirmed by high performance liquid chromatography analysis and gas chromatography-mass spectroscopy analysis respectively.The desulfurization kinetics revealed that DMT-7 started desulfurization of DBT into 2HBP after the lag phase of 24 hr,exponentially increasing the accumulation of 2HBP up to 15 days leading to approximately 60% desulfurization of the DBT.However,further growth resulted into DBT degradation.The induced culture of DMT-7 showed shorter lag phase of 6 hr and early onset of stationary phase within 10 days for desulfurization as compared to that of non-induced culture clearly indicating the inducibility of the desulfurization pathway of DMT-7.In addition,Lysinibacillus sphaericus DMT-7 also possess the ability to utilize broad range of substrates as sole source of sulfur such as benzothiophene,3,4-benzo DBT,4,6-dimethyl DBT,and 4,6-dibutyl DBT.Therefore,Lysinibacillus sphaericus DMT-7 could serve as model system for efficient biodesulfurization of diesel and petrol.

Effect of lithium on growth of bone marrow stromal cells

Objective To investigate the effect of lithium on the growth of bone marrow stromal cells Methods We compared the different effects of lithium with the concentration of 0 1-12mmol/L on the growth of murine fibroblast colony forming unit (mCFU F), human fibroblast colony forming unit (hCFU F) and their intercolonial cells Pure murine bone marrow stromal cell assay was used to investigate the mechanism Results Cultures with 1-4mmol/L lithium resulted in significant increase in the number of hCFU F, but distinct decrease in the number of mCFU F Furthermore, there were much more endothelial cells between mCFU Fs than those between hCFU Fs The direct effect of 1 4mmol/L lithium on the growth of pure murine fibroblasts and endothelial cells was stimulatory Serum free endothelial cell conditioned medium (EC CM) could inhibit the growth of mCFU F Conclusions 1-4mmol/L lithium has stimulatory effect on the growth of pure murine fibroblasts and endothelial cells The inhibitory effect of lithium on mCFU F formation may result from the effect of increased endothelial cells