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Aim Hepatocytes can proliferate and regenerate when injured by toxins, viral infections, and so on. Augmenter of liver regeneration (ALR) is a key regulator of liver regeneration, but the mechanism is unknown. The role of ALR in other cell types is not known. In the present study, we investigated the relationship between microRNA (miRNA)-26a and ALR in the Huh7 cell line and adipose tissue-derived mesenchymal cells from chronic liver disease patients and healthy individuals. Methods Huh7 cells were transfected independently with ALR and miRNA-26a expression vectors, and their effects on cell proliferation, the expression of miRNA-26a, and activation of the phosphatase and tensin homolog and Akt signaling pathways were determined. The experiments were repeated on mesenchymal stem cells derived from healthy individuals and chronic liver disease patients to see whether the observations can be replicated in primary cells. Results Overexpression of ALR or miRNA-26a resulted in an increase of the phosphorylation of Akt and cyclin D1 expression, whereas it resulted in decreased levels of p-GSK-3β and phosphatase and tensin homolog in Huh7 cells. The inhibition of ALR expression by ALR siRNA or anti-miR-26a decreased the Akt/cyclin D1 signaling pathway, leading to decreased proliferation. Mesenchymal stem cells isolated from the chronic liver disease patients had a higher ALR expression, while the mesenchymal stem cells isolated from healthy volunteers responded to the growth factor treatments for increased ALR expression. It was found that there was a significant increase in miRNA-26a expression and proliferation. Conclusions These data clearly showed that ALR induced the expression of miRNA-26a, which downregulated phosphatase and tensin homolog, resulting in an increased p-Akt/cyclin D1 pathway and enhanced proliferation in hepatic cells.

Generation of human embryonic stem cells and elucidation of method to generate induced pluripotent stem cells (iPSCs) were major milestones in the history of regenerative medicine. Since then, several years have passed; however, no major therapies came out of them. They are not good enough yet for clinics. An important reason is the epigenetic and genetic deviations. With recent progress in somatic cell nuclear transfer (SCNT), we can generate human diploid embryonic stem cells. SCNT generates pluripotent stem cells much faster than iPSC technique. However, the process is highly inefficient and expensive. Probably because the reprogramming process is too quick, cells get lesser time to adjust and evolve the epigenetic landscape, they suffer from epigenetic defects. Combining iPSC technology with SCNT could be a solution. That is, by using the nucleus of the candidate somatic cell which was reprogrammed to pluripotent state through the iPSC technology would generate therapeutic quality pluripotent stem cells and this new technique may be called induced pluripotent stem cell nuclear transfer (iPCNT). Improvements in this direction could facilitate therapeutic and reproductive cloning, a concept which is considered unethical today. However, ethics is a function of time, place, and people and therefore not inviolate.

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

BackgroundGenomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the site which is disrupted upon integration of Adeno Associated Virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in Progressive Familial Intra Hepatic Cholestasis Type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient.MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines.ResultsWe found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA.ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract).Pictorial AbstractABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5’ and 3’ overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2/2019-nCoV) infection is an emerging pandemic. The virus binds to angiotensin converting enzyme 2 (ACE2) and TMPRSS2 which are abundantly expressed on various human cells including lung epithelial cells and intestinal cells and the virus can infect these cells. Currently no specific treatments or vaccines are available for this disease. A per oral live attenuated vaccine can be beneficial in SARS-Cov-2 infection because the attenuated virus initially infects the gut, stimulates the mucosa associated immune system sparing the respiratory system during the initial immune response. The live virus can also spread in the community boosting herd immunity.

Artificial Intelligence (AI) and access to "Big Data" together with the evolving techniques in biotechnology will change the medical practice a big way. Many diseases such as type II diabetes will no longer be considered as a single disease. Many familiar cancers such as cancer of liver or pancreas will have hundreds of subtypes whose management will be very different. The way we think about diseases will change. It will no longer be possible for clinicians to make a diagnosis, remember the names of diseases, the names of drugs or management protocols without the help of computers. As computer intelligence becomes more important than human intelligence in deciding diagnosis and treatment there will be a paradigm in the role of doctors. Internet, computers and social media will become more important than individuals in decision making. As a result, medicine will go more and more egalitarian ("wiki") with increasing community participation in health decision making and management. A socialistic pattern will evolve over time globally as an adaptive reaction to the pressures put by artificial intelligence. This is because the individual differences in knowledge or intellect between human beings will become less apparent compared to the super powers of artificial intelligence. Qualities which are unique for humans such as compassion, empathy and emotional care will decide the professional success of future physicians even more than today. Today we are using artificial intelligence in diagnosis and prediction to help clinicians. Clinical algorithms and human experience cannot be replaced by machines. It will take many years to completely merge or replace humans with machines. However, we need to modify our medical education system in order to prepare the medical community and sensitize the society well in advance for a smooth transition.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

Escherichia coli are commensal gastrointestinal microflora of humans, but few strains may cause food-borne diseases. Present study aimed to identify antimicrobial resistant (AMR), biofilm-forming E. coli from Indian dairy and meat products. A total of 32 E. coli isolates were identified and evaluated for biofilm-formation. EMC17, an E. coli isolate was established as a powerful biofilm-former that attained maximum biofilm-formation within 96 h on glass and stainless-steel surfaces. Presence and expression of virulence-associated genes (adhesins, invasins and polysaccharides) and ability to adhere and invade human liver carcinoma HepG2 cell lines implicates EMC17 to be pathotype belonging to Extra-intestinal Pathogenic E. coli (ExPEC). Antibiotic profiling of EMC17 identified it as multi-drug resistant (MDR) strain, possessing extended spectrum β-lactamases (ESBL's) and biofilm phenotype. Early production of quorum sensing molecules (AHLs) alongside EPS production facilitated early onset of biofilm formation by EMC17. Furthermore, the biofilm-forming genes of EMC17 were significantly upregulated 3–27 folds in the biofilm-state. This study showed prevalence of MDR, biofilm-forming, pathogenic E. coli in Indian dairy and meat products that potentially serve as reservoirs for transmission of antimicrobial-resistant (AMR) genes of bacteria from food to humans and pose serious food safety threat.

ObjectiveTo study the role of miRNA-181a and augmenter of liver regeneration in TGF-β-induced fibrosis in hepatic stellate cells.MethodsLX2 cells were treated with 20 ng/ml TGF-β for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-β receptor II (TGFβ-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and β-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA.ResultsTGF-β induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFβ-RII and increased expression E-cadherin.ConclusionTGF-β induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-β action by decreasing the expression of TGFβ-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.

Nonalcoholic fatty liver disease (NAFLD), a common cause of cryptogenic cirrhosis is often associated with metabolic syndrome (MS) in the West. However, its association with MS in the Indian population is not well studied.To evaluate the association NAFLD with MS using the modified ATP-III criteria.Seventy-six (62 men, mean age 40.05±11.4 years, range 18-66) apparently healthy subjects with fatty liver and histological evidence of NAFLD; with (64.5%) or without raised ALT, and 100 healthy controls were included in the study. The anthropometric measurements, metabolic parameters, tests of liver function and liver histology were studied.Twenty-one percent of the patients compared to 8% controls (p0.01) had associated MS; according to ATP-III criteria while 42 and 12% were affected when the modified ATP-III criteria were used. About 35% of non-diabetic patients were insulin resistant with homeostatic model assessment-insulin resistance (HOMA-IR) cut-off set at 3. In patients, compared to controls, the mean BMI (25.2 vs. 22.7, p0.01) and waist circumferences (92.9 cm vs. 80.8 cm, p0.01) were higher. Seventy-nine percent of the patients and 44% of the controls were over weight. Stage 1 fibrosis was seen in 30 (39.5%), stage 2 in 10 (13.2%), stage 3 in 6 (7.9%) and stage 4 in 13 (17%) patients.Insulin resistance and obesity are associated in a proportion of Indian patients with NAFLD. However, the association with MS as defined by ATP-III in Indian patients is not strong compared to the West. It is likely that pathogenetic mechanisms unrelated to MS underlie development of NAFLD in a proportion of Indian patients.

Background Asian Indians are known to be more insulin resistant for the same degree of weight gain. It is therefore likely that the adipokine profile in nonalcoholic fatty liver disease (NAFLD) in Asian Indian population could be different from the Western subjects. Aims To study the serum adiponectin, resistin, leptin and TNF-α profile in NAFLD and cryptogenic cirrhosis patients. Subjects and methods Body mass indices, insulin resistance and serum adipokine levels were studied in 56 patients; 10 with fatty liver, 30 with nonalcoholic steatohepatitis (NASH) and 16 with cryptogenic cirrhosis. Eighteen healthy controls were also included. Results Patients in general were obese compared to controls (mean BMI 26.9 ± 4.5 vs. 22.6 ± 2.5, respectively, p p = 0.037). Insulin Resistance as assessed by homeostasis model assessment (HOMA) was higher in obese than lean, NAFLD patients (HOMA IR obese, median = 2.8, range = 0.8–16.3 and lean: median = 1.05, range = 0.51–2.75, p = 0.003). Lean NAFLD patients had adiponectin levels lower than obese patients (3 ± 1 μg/ml vs.6.7 ± 3.8 μg/ml respectively, p = 0.003). Serum resistin levels were higher in NAFLD patients (3.7 ± 3 ng/ml) than controls (2.1 ± 1.7 ng/ml, p = 0.007). This difference was significant even when cirrhotic patients were excluded (3.4 ± 2.7 ng/ml, p = 0.036). Serum leptin levels were raised in cryptogentic cirrhosis compared to NASH ( p = 0.03). All adipokines tested were raised in cirrhotic patients compared to NAFLD and controls. Conclusions A significant reduction in serum adiponectin and increase in serum resistin levels were observed in NAFLD patients, more so in lean than obese NAFLD. This paradoxical decrease of serum adiponectin as well as low frequency of insulin resistance in lean NAFLD suggests a possible different etiology for this subset of patients.

Gestational diabetes mellitus (GDM) is a common public health issue of pregnancy and women who have had GDM are at high risk for developing of diabetes mellitus Type-2. The aim of this study was to find the association between various clinical and biochemical parameters and GDM. One hundred and six consecutive patients who attended the out patient unit of department of gynecology, Kottayam Medical College, were enrolled in the study and followed up through the whole antenatal, intra-partum and post-partum periods to identify the obstetric outcome. We found that the prevalence of GDM was seven times higher in those with higher waist-hip ratio (WHR0.85) compared with those having a lower WHR (p0.001).Those with higher WHR gained more weight than other group (10.6 kg vs. 8.1 kg; p0.001). Obesity (BMIor =23) and higher WHR were associated with increased risk of gestational diabetes (BMIor =23: OR = 7.5, CI 95% = (1.61-34.31), p = 0.013; WHR0.85: OR = 12.05, CI 95% = (1.82-77.43), p = 0.007). We found that a WHR of 0.849 has the optimal sensitivity and specificity for the prediction of GDM. A waist circumference of 85.5 cm (with sensitivity of 75%, specificity 81.4%) and a BMI of 24.3 kg/m(2) (sensitivity 75%, specificity 86.5%) had the best predictive value. In conclusion, we found that maternal obesity has a strong correlation with obstetric complications. We found WHR is more important risk determinant for GDM in overweight/obese women than women with normal weight/lean.

Developments in cell therapy and organ transplantation would transform personalized medicine as perceived today. Adult stem cells and induced pluripotent stem cell (iPSC) derivatives can be used to repair, rejuvenate, or replace damaged organs. Transplanted cells act by secreted factors, cell-cell interaction, and immune modulation or by repopulating the damaged tissue. Repopulating organs with genetically corrected progenitor cells or differentiated cells could correct several rare genetic diseases at a very personal level. Cell therapy has applications in curing infections as well from life-threatening fungal infections to HIV. Today tools such as zinc finger nucleases and TALENS make it possible to manipulate human genome precisely. In the future transplantable personalized whole organs will be generated using iPSC and tetraploid complementation, and techniques which are still in infancy. Cells and organs will be engineered and standardized to be compatible with a wide range of drugs and environmental conditions or become more personalized for special needs. Three dimensional printing technologies can now generate simple organs like urinary bladder in a personalized way, but in the future, it might be possible to “print” more complex organs. Genetically engineered cells would play a major role in the future of fighting cancer in a personalized manner. Stem cell biology, genetic engineering, and regenerative medicine catapulted by latest developments in basic sciences would revolutionize human life, and we need to prepare and sensitize our society well ahead.

Potential of nonantibiotic therapies for treatment of Helicobacter pylori-related acid peptic disease remains underexplored. Several clinical studies have shown that higher prevalence of H. pylori infection is associated with low Vitamin C (Vit C) level in serum and gastric juice. However, there is no consensus regarding the usefulness of Vit C supplementation in the management of H. pylori infection. Surveying the existing literature we conclude that high concentration of Vit C in gastric juice might inactivate H. pylori urease, the key enzyme for the pathogen's survival and colonization into acidic stomach. Once infection established, urease is not very important for its survival. The role of Vit-C as anti-H. pylori agent in peptic ulcer diseases appears to be preventive rather than curative. Rather than supplementing high dose of Vit C along with conventional triple therapy, it is preferable to complete the conventional therapy and thereafter start Vit C supplementation for extended period which would prevent reinfection in susceptible individuals, provided the patients are not achlorhydric. Further studies are required to prove the role of Vit C in susceptible population.

Adipose tissue is critical in energy homeostasis. Adipose tissue 'buffers' the lipids and energy rich compounds which are pumped into the blood stream soon after meals. It senses, signals other organs like liver and brain about the energy reserves via adipokines. Adiponectin, the most abundant adipokine has insulin sensitizing, anti-inflammatory antiatherogenic and antisteatotic effects. Adipose tissue dysfunction is accompanied by abnormal lipid distribution and storage which contributes to diseases like diabetes, nonalcoholic fatty liver disease and atherosclerosis. Obesity and lipodystrophy are associated with dysfunctional adipocytes. Pre-adipocytes are easy to isolate and culture. A personalized depot specific liposuction to remove the inactive adipocytes followed by adipocyte repopulation could be useful in the treatment of these diseases.

Nonalcoholic fatty liver disease (NAFLD) is a group of diseases with excess fat in liver in the absence of a poorly defined limit of alcohol consumption. Most common variety, a universal public health problem, is associated with insulin resistance caused by a host of genetic and epigenetic defects modulated by life style and environmental factors. In fact the term NAFLD is loose to incorporate so many etiologies except alcoholism and few other etiologies, presenting as fat in liver. However as a sign fatty liver is very important in predicting the risk of diabetes, cardiovascular disease, stroke, cirrhosis and cancer. Abnormal fat accumulation can result from several defects in nuclear receptors associated with lipid sensing, synthesis and oxidation like LXR, FXR, SREBP, ChREBP and PPAR; defects in the lipid influx-efflux channels, insulin signaling, proteins involved in fatty acid catabolism, defects in adipose tissue development and function, inappropriate nutrition and finally defects in neural regulatory mechanisms. The progress of the disease is determined by the basic defects which results in fat accumulation, an individual’s immunological response to the accumulated fat and its derivatives and the oxidant stress response. Congregation of unrelated genetic defects under same diagnosis ‘NAFLD’ can result in inefficient patient management. Further studies are required to understand the molecular basis of fatty liver to enable a personalized management of diseases presenting as fatty liver in the absence of alcohol abuse.

Strategies to fill the huge gap in supply versus demand of human organs include bioartificial organs, growing humanized organs in animals, cell therapy, and implantable bioengineered constructs. Reproducing the complex relations between different cell types, generation of adequate vasculature, and immunological complications are road blocks in generation of bioengineered organs, while immunological complications limit the use of humanized organs produced in animals. Recent developments in induced pluripotent stem cell (iPSC) biology offer a possibility of generating human, patient-specific organs in non-human primates (NHP) using patient-derived iPSC and NHP-derived iPSC lacking the critical developmental genes for the organ of interest complementing a NHP tetraploid embryo. The organ derived in this way will have the same human leukocyte antigen (HLA) profile as the patient. This approach can be curative in genetic disorders as this offers the possibility of gene manipulation and correction of the patient's genome at the iPSC stage before tetraploid complementation. The process of generation of patient-specific organs such as the liver in this way has the great advantage of making use of the natural signaling cascades in the natural milieu probably resulting in organs of great quality for transplantation. However, the inexorable scientific developments in this direction involve several social issues and hence we need to educate and prepare society in advance to accept the revolutionary consequences, good, bad and ugly.

Fatty liver is present in over ten percentage of the world population and it is a growing public health problem. Nonalcoholic fatty liver disease (NAFLD) is not a single disease, but encompasses a spectrum of diseases of different etiologies. It is difficult to find highly specific and sensitive diagnostic biomarkers when a disease is very complex. Therefore, we should aim to find relevant prognostic markers rather than accurate diagnostic markers which will help to minimize the frequency of liver biopsies to evaluate disease progression. There are several biomarker panels commercially available, however, there is no clear evidence that more sophisticated panels are better compared to simple criteria such as, presence of diabetes over five years, metabolic syndrome, obesity, obstructive sleep apnea, aspartate transaminase/alanine transaminase (ALT) ratio > 0.8 or ferritin levels > 1.5 times normal in patients with over six month history of raised ALT and/or ultrasonological evidence of fat in the liver. Currently the biomarker panels are not a replacement for a liver biopsy. However the need and benefit of liver biopsy in NAFLD is questionable because there is no convincing evidence that biopsy and detailed staging of NAFLD improves the management of NAFLD and benefits the patient. After all there is no evidence based treatment for NAFLD other than management of lifestyle and components of “metabolic syndrome”.