Discovery of Zika Virus Dependency and Restriction Factors Using Flow-Based Arrayed CRISPR Screening for Identification of Targets (FACS-IT).

The discovery and implementation of CRISPR/Cas9 tools in pooled genetic screens have allowed for the rapid, high-fidelity identification of host-virus interactions. However, pooled CRISPR screening has significant limitations in its ability both to perform cell biology and plate reader-based screens and to find alleles that result in intermediate-strength phenotypes. Here we introduce an arrayed CRISPR screening method, FACS-IT, which allows researchers to use high content imaging analysis, plate reader assays, cell supernatant characterization, and percent infectivity to characterize CRISPR-mediated gene disruptions causing both moderate and extreme phenotypic changes. By using flow sorting capabilities and CRISPR libraries that are widely available, FACS-IT overcomes both the significant limitation of pooled screening approaches and the prohibitive costs of large-scale arrayed CRISPR reagents. In doing so, FACS-IT will enable researchers to creatively use CRISPR screening to obtain a deeper understanding of biology across a wide range of fields and applications.

Prevalence of excessive internet use and its correlation with associated psychopathology in 11th and 12th grade students.

BACKGROUND: Globally, the number of internet users has crossed the three-billion mark, while in India users grew over 17% in the first 6 months of 2015 to 354 million. This study presented a background on internet use and the existence of excessive internet use. AIM: To study the extent of internet use in 11th and 12 grade students and the psychopathology, if any, associated with excessive internet use. METHODS: 426 students who met the inclusion criteria were recruited from 11th and 12th grade classes from Kendriya Vidyalaya, New Delhi, India, and were assessed by Young's Internet Addiction Test and the Strength and Difficulties Questionnaire. RESULTS: Among the 426 students, the mean internet addiction total score was 36.63 (20.78), which indicated mild level of internet addiction. 1.41% (six students) was diagnosed as excessive internet users, while 30.28% and 23.94% were classified as moderate and mild internet users, respectively. The prevalence of internet addiction between gender was 58.22% in males and 41.78% in females. While both positive (prosocial) and negative (hyperactivity, emotional, conduct and peer problem) impacts of internet use were reported by students, in the current study excessive use of internet had a negative impact on students' lives as compared with positive impact, which was statistically significant (p<0.0001). CONCLUSION: Excessive internet use led to abnormal behaviours which cause negative consequences to users. Early diagnosis of risk factors related to excessive internet use, provides education about responsible use and supervision of students by family members.

HBeAg-induced miR-106b promotes cell growth by targeting the retinoblastoma gene.

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC). The association between hepatitis B "e" antigen (HBeAg) and HCC is well-established by epidemiological studies. Nonetheless, the biological role of HBeAg in HCC remains enigmatic. We investigate the role of HBeAg in HBV-related HCC. Our findings suggest that HBeAg enhances cell proliferation and accelerates progression from G0/G1 phase to the S phase of the cell cycle in Huh7 cells. Examination of host gene expression and miRNA expression profiles reveals a total of 21 host genes and 12 host miRNAs that were differentially regulated in cells expressing HBeAg. Importantly, HBeAg induced the expression of miR-106b, an oncogenic miRNA. Interestingly, HBeAg-expression results in a significant reduction in the expression of retinoblastoma (Rb) gene, an experimentally validated target of miR-106b. Inhibition of miR-106b significantly increased the expression of the Rb gene, resulting in reduced cell proliferation and slowing of cell cycle progression from the G0/G1 phase to S phase. These observations suggest that the up-regulation of miR-106b by HBeAg contributes to the pathogenesis of HBV-related HCC by down-regulating the Rb gene. Our results highlight a role for HBeAg in HCC and provide a novel perspective on the molecular mechanisms underlying HBV-related HCC.

A G-quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in HBV genotype B.

HBV genotypes differ in pathogenicity. In addition, genotype-specific differences in the regulation of transcription and virus replication exist in HBV, but the underlying mechanisms are unknown. Here, we show the presence of a G-quadruplex motif in the promoter of the preS2/S gene; this G-quadruplex is highly conserved only in HBV genotype B but not in other HBV genotypes. We demonstrate that this G-quadruplex motif forms a hybrid intramolecular G-quadruplex structure. Interestingly, mutations disrupting the G-quadruplex in HBV genotype B reduced the preS2/S promoter activity, leading to reduced hepatitis B surface antigen (HBsAg) levels. G-quadruplex ligands stabilized the G-quadruplex in genotype B and enhanced the preS2/S promoter activity. Furthermore, mutations disrupting the G-quadruplex in the full-length HBV genotype B constructs were associated with impaired virion secretion. In contrast to typical G-quadruplexes within promoters which are negative regulators of transcription the G-quadruplex in the preS2/S promoter of HBV represents an unconventional positive regulatory element. Our findings highlight (a) G-quadruplex mediated enhancement of transcription and virion secretion in HBV and (b) a yet unknown role for DNA secondary structures in complex genotype-specific regulatory mechanisms in virus genomes.

Genome-wide analysis of G-quadruplexes in herpesvirus genomes.

BACKGROUND: G-quadruplexes are increasingly recognized as regulatory elements in human, animal, bacterial and plant genomes. The presence and function of G-quadruplexes are not well studied among herpesviruses; in particular, there are no systematic genome-wide analysis of these important secondary structures in herpesvirus genomes. RESULTS: We performed genome-wide analysis of putative quadruplex sequences (PQS) in human herpesviruses. We found unusually high PQS densities among human herpesviruses. PQS are enriched in the repeat regions and regulatory regions of human herpesviruses. Interestingly, PQS densities are higher in regulatory regions of immediate early genes compared to early and late genes in most herpesviruses. In addition, the majority of genes functionally conserved across human herpesviruses contain one or more PQS within the regulatory regions. We also describe the existence of unique intramolecular PQS repeats or repetitive G-quadruplex motifs in herpesviruses. Functional studies confirm a role for G-quadruplexes in regulating the gene expression of human herpesviruses. CONCLUSION: The pervasiveness of PQS, their enrichment and conservation at specific genomic locations suggest that these structural entities may represent a novel class of functional elements in herpesviruses. Our findings provide the necessary framework for studies on the biological role of G-quadruplexes in herpesviruses.

Case report of comorbid schizophrenia and obsessive compulsive disorder in a patient who was tube-fed for four years by family members because of his refusal to eat.

Refusal to eat is a common presentation in many psychiatric disorders including obsessive compulsive disorder and schizophrenia. In the acute situation it may be a medical emergency; when it becomes chronic it can become an ingrained behavior that is difficult to change. The diagnosis of individuals who refuse to eat may be difficult, particularly in persons with comorbid medical problems, impaired intelligence, or lack of insight into their condition. Tube-feeding is an effective short-term intervention that can be discontinued when the patient re-starts oral intake. However, in some situations patients may become dependent on the use of tube-feeding. We present a case report of a patient with schizophrenia, obsessive compulsive disorder, borderline intelligence, and seizure disorder who was tube-fed by his family members for more than three years because he refused to eat orally.

Enhanced hepatitis B virus (HBV) pre-genomic RNA levels and higher transcription efficiency of defective HBV genomes.

Defective hepatitis B virus (dHBV) particles contain genomes corresponding to singly spliced HBV RNA. A limited number of studies show that dHBV is present in all chronically HBV-infected patients. Clinical studies have linked dHBV and dHBV gene products to high virus loads and liver damage. The replication characteristics of dHBV genomes remain poorly understood. We found that the splice donor/acceptor sites critical for the formation of dHBV genomes are conserved across HBV genotypes. We report a novel method to create dHBV constructs from corresponding wild-type (WT) HBV constructs. We assessed the transcriptional characteristics of the dHBV constructs with those of the corresponding WT construct using a cell culture model. Interestingly, dHBV constructs had higher pre-genomic RNA levels, transcription efficiency, HBV e antigen levels and intracellular HBV core antigen levels compared with the corresponding WT HBV constructs. Our findings highlight previously unrecognized fundamental molecular characteristics of dHBV genomes and their potential role in the pathogenesis of HBV infection.

Hepatitis B "e" antigen-mediated inhibition of HBV replication fitness and transcription efficiency in vitro.

A mutation at nucleotide 1896 (G1896A) is the most common cause for the loss of HBeAg. In contrast to clinical data, cell culture studies report a high-replicating phenotype for the G1896A mutant. Differences between the wild-type and the G1896A mutant in early steps of HBV replication including the synthesis of pre-genomic RNA and transcripts have not been investigated. The G1896A mutant is associated with higher replication fitness, transcription efficiency and higher levels of secreted HBsAg than the wild-type. Interestingly, trans-complementation of the G1896A mutant with HBeAg lowers the replication fitness and transcriptionefficiency to levels comparable to that of the wild-type. Our results highlight the role of HBeAg in modulating the early steps in HBV replication. In sum, our findings highlight the role of HBeAg in regulating hepatitis B virus replication fitness and transcription efficiency and new insights on the early steps of replication in the G1896A mutant.

Sleeping beauty: kleine-levin syndrome.

Kleine-Levin syndrome (KLS) alias sleeping beauty syndrome, is a rare sleep disorder. Clinically presenting as episodes of hypersomnolence, behavioral and cognitive disturbances, hyperphagia and hypersexuality. KLS may have an idiopathic onset or may be precipitated by neurological event or infection. Until date, no definite underlying cause is established and neither there are any definitive management guidelines. It remains a diagnosis of exclusion after other psychiatric and neurological causes have been ruled out. Coloring of presentation with behavioral and mood elements makes it important for a psychiatrist to be well-informed about the condition to avoid the erroneous diagnosis. KLS is a devastating illness, which robs the patient of time, experiences, and relationships. An early diagnosis and effective management can help patient escape from the morbidity caused by this disorder. Armodafinil and oxcarbamazepine have found to be effective in two of the case. The emphasis of this report is to add to the existing clinical knowledge of neurologists, psychiatrists and physicians. In the future, research is needed on genetic etiology and management of this disorder.

Mistaken gender identity in non-classical congenital adrenal hyperplasia.

Gender identity is the sense of belonging that one feels for a particular sex psychologically and socially, independent of one's biological sex. There is much less systematic data on gender identity in females with congenital adrenal hyperplasia (CAH). We report a case of non-classical CAH presenting as a case of gender identity disorder.

CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

The Warburg effect: insights from the past decade.

Several decades ago, Otto Warburg discovered that cancer cells produce energy predominantly by glycolysis; a phenomenon now termed "Warburg effect". Warburg linked mitochondrial respiratory defects in cancer cells to aerobic glycolysis; this theory of his gradually lost its importance with the lack of conclusive evidence confirming the presence of mitochondrial defects in cancer cells. Scientists began to believe that this altered mechanism of energy production in cancer cells was more of an effect than the cause. More than 50 years later, the clinical use of FDG-PET imaging in the diagnosis and monitoring of cancers rekindled the interest of the scientific community in Warburg's hypothesis. In the last ten years considerable progress in the field has advanced our understanding of the Warburg effect. However, it still remains unclear if the Warburg effect plays a causal role in cancers or it is an epiphenomenon in tumorigenesis. In this review we aim to discuss the molecular mechanisms associated with the Warburg effect with emphasis on recent advances in the field including the role of epigenetic changes, miRNAs and post-translational modification of proteins. In addition, we also discuss emerging therapeutic strategies that target the dependence of cancer cells on altered energy processing through aerobic glycolysis.

Molecular mechanisms underlying occult hepatitis B virus infection.

Chronic hepatitis B virus (HBV) infection is a complex clinical entity frequently associated with cirrhosis and hepatocellular carcinoma (HCC). The persistence of HBV genomes in the absence of detectable surface antigenemia is termed occult HBV infection. Mutations in the surface gene rendering HBsAg undetectable by commercial assays and inhibition of HBV by suppression of viral replication and viral proteins represent two fundamentally different mechanisms that lead to occult HBV infections. The molecular mechanisms underlying occult HBV infections, including recently identified mechanisms associated with the suppression of HBV replication and inhibition of HBV proteins, are reviewed in detail. The availability of highly sensitive molecular methods has led to increased detection of occult HBV infections in various clinical settings. The clinical relevance of occult HBV infection and the utility of appropriate diagnostic methods to detect occult HBV infection are discussed. The need for specific guidelines on the diagnosis and management of occult HBV infection is being increasingly recognized; the aspects of mechanistic studies that warrant further investigation are discussed in the final section.