Existence of Replication-Competent Minor Variants with Different Coreceptor Usage in Plasma from HIV-1-Infected Individuals.

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations.IMPORTANCE The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.

Anti-Melanogenesis Activity of 6-O-Isobutyrylbritannilactone from Inula britannica on B16F10 Melanocytes and In Vivo Zebrafish Models.

A potential natural melanogenesis inhibitor was discovered in the form of a sesquiterpene isolated from the flowers of Inula britannica, specifically 6-O-isobutyrylbritannilactone (IBL). We evaluated the antimelanogenesis effects of IBL on B16F10 melanocytes and zebrafish embryos. As a result, we found that 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production was reduced in a dose-dependent manner in B16F10 cells by IBL. We also analyzed B16F10 cells that were and were not treated with IBMX, investigating the melanin concentration, tyrosinase activity, mRNA levels. We also studied the protein expressions of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related proteins (TRP1, and TRP2). Furthermore, we found that melanin synthesis and tyrosinase expression were also inhibited by IBL through the modulation of the following signaling pathways: ERK, phosphoinositide 3-kinase (PI3K)/AKT, and CREB. In addition, we studied antimelanogenic activity using zebrafish embryos and found that the embryos had significantly reduced pigmentation in the IBL-treated specimens compared to the untreated controls.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

1-Methoxylespeflorin G11 Protects HT22 Cells from Glutamate-Induced Cell Death through Inhibition of ROS Production and Apoptosis.

This study aimed to investigate the neuroprotective effects of 1-methoxylespeflorin G11 (MLG), a pterocarpan, against glutamate-induced neurotoxicity in neuronal HT22 hippocampal cells. The protective effects of MLG were evaluated using MTT assay and microscopic analysis. The extent of apoptosis was studied using flow cytometric analysis performed on the damaged cells probed with annexin V/propidium iodide. Moreover, mitochondrial reactive oxygen species (ROS) were assessed using flow cytometry through MitoSOXTM Red staining. To determine mitochondrial membrane potential, staining with tetramethylrhodamine and JC-1 was performed followed by flow cytometry. The results demonstrated that MLG attenuates glutamate-induced apoptosis in HT22 cells by inhibiting intracellular ROS generation and mitochondrial dysfunction. Additionally, MLG prevented glutamate-induced apoptotic pathway in HT22 cells through upregulation of Bcl-2 and downregulation of cleaved PARP-1, AIF, and phosphorylated MAPK cascades. In addition, MLG treatment induced HO-1 expression in HT22 cells. These results suggested that MLG exhibits neuroprotective effects against glutamate-induced neurotoxicity in neuronal HT22 cells by inhibiting oxidative stress and apoptosis.

Brunner's gland hyperplasia associated with lipomatous pseudohypertrophy of the pancreas presenting with gastrointestinal bleeding: A case report.

BACKGROUND: Brunner's gland hyperplasia (BGH) is a rare benign lesion of the duodenum. Lipomatous pseudohypertrophy (LiPH) of the pancreas is an extremely rare disease. Because each condition is rare, the probability of purely coincidental coexistence of both conditions is extremely low. CASE SUMMARY: We report a 26-year-old man presenting to our hospital with symptoms of recurrent upper gastrointestinal bleeding. Upper gastrointestinal endoscopy showed a huge pedunculated polypoid lesion in the duodenum with bleeding at the base of the lesion. Histopathological examination of the duodenal biopsy specimens showed BGH. Besides, abdominal computed tomography and magnetic resonance imaging revealed marked fat replacement over the entire pancreas, confirmed by histopathological evaluation on percutaneous pancreatic biopsies. Based on the radiological and histological findings, LiPH of the pancreas and BGH were diagnosed. The patient refused any surgical intervention. Therefore, he was managed with supportive treatment. The patient's symptoms improved and there was no further bleeding. CONCLUSION: This is the first well-documented case showing the coexistence of LiPH of the pancreas and BGH.

Apoptosis in Leukemic Cells Induced by Anti-proliferative Coumarin Isolated from the Stem Bark of Fraxinus rhynchophylla.

Esculetin 6-O-ß-D-arabinofuranosyl-(1→6)-ß-D-glucopyranoside (EAG) is a coumarin glycoside isolated from the stem bark of Fraxinus rhynchophylla. This study scrutinized the anti-proliferative activity of EAG on blood cancer-derived Jurkat leukemic cells. Cell viability assays in leukemic cancer cells determined that EAG possesses potent anti-proliferative effects. Moreover, treatment with EAG increased the proportion of apoptotic cells, resulted in cell cycle arrest being induced at the subG0/ G1 phase, and reduced the proportion of cells present in the S phase. In addition, mitochondrial membrane potential was reduced by EAG in Jurkat cells. Additionally, EAG triggered apoptosis that was mediated by the downregulation of BCL-XL, p-IκBα, and p-p65 expressions in addition to the upregulation of cleaved Caspase 3 and BAX expressions. These findings revealed that the toxic effect of EAG was mediated by intracellular signal transduction pathways that involved a mechanism in which reactive oxygen species (ROS) were upregulated. Thus, this study concludes that EAG could potentially serve as a therapeutic agent for leukemia.

Coxsackieviruses A6 and A16 associated with hand, foot, and mouth disease in Vietnam, 2008-2017: Essential information for rational vaccine design.

Development of multivalent hand, foot, and mouth disease (HFMD) vaccines against enterovirus A71 (EV-A71) and several non-EV-A71 enteroviruses is needed for this life-threatening disease with a huge economic burden in Asia-Pacific countries. Comprehensive studies on the molecular epidemiology and genetic and antigenic characterization of major causative enteroviruses will provide information for rational vaccine design. Compared with molecular studies on EV-A71, that for non-EV-A71 enteroviruses remain few and limited in Vietnam. Therefore, we conducted a 10-year study on the circulation and genetic characterization of coxsackievirus A16 (CV-A16) and CV-A6 isolated from patients with HFMD in Northern Vietnam between 2008 and 2017. Enteroviruses were detected in 2228 of 3212 enrolled patients. Of the 42 serotypes assigned, 28.4% and 22.4% accounted for CV-A6 and CV-A16, being the second and the third dominant serotypes after EV-A71 (31.7%), respectively. The circulation of CV-A16 and CV-A6 showed a wide geographic distribution and distinct periodicity. Phylogenetic analyses revealed that the majority of Vietnamese CV-A6 and CV-A16 strains were located within the largest sub-genotypes or sub-genogroups. These comprised strains isolated from patients with HFMD worldwide during the past decade and the Vietnamese strains have been evolving in a manner similar to the strains circulating worldwide. Amino acid sequences of the putative functional loops on VP1 and other VPs among Vietnamese CV-A6 and CV-A16 isolates were highly conserved. Moreover, the functional loop patterns of VP1 were similar to the dominant patterns found worldwide, except for the T164K substitution on the EF loop in Vietnamese CV-A16. The findings suggest that the development of a universal HFMD vaccine, at least in Vietnam, must target CV-A6 and CV-A16 as two of the three major HFMD-causing serotypes. Vietnamese isolates or their genome sequences can be considered for rational vaccine design.

Submandibular neurilemmoma; a diagnostic dilemma.

Neurilemmomas are slow growing, benign neoplasms of neural crest Schwann cell origin. They arise from any peripheral, spinal or cranial nerve except the olfactory and optic. Presentation is usually asymptomatic but focal neurological signs and symptoms may be associated with nerve compression. With approximately one third of all documented cases presenting in the head and neck region, we report a case of a submandibular neurilemmoma misdiagnosed pre-operatively. The diagnostic difficulties are discussed and the current literature reviewed. This case highlights the importance of inclusion of nerve sheath tumours in differential diagnoses of soft tissue lesions in the head and neck.

'Hit by the wind' and temperature-shift panic among Vietnamese refugees.

Surveying 60 Vietnamese patients with either current or past post-traumatic stress disorder, this article aims to phenomenologically characterize the syndrome of 'hit by the wind' in a multidimensional manner. This includes determining the patient conceptualization of the disorder, profiling 'hit by the wind' episodes suffered by patients in the previous month, and presenting case vignettes. Eighteen of the 60 patients (30%) suffered at least one episode of 'hit by the wind' in the last month; all 18 patients had at least one episode of 'hit by the wind' in the last month that met panic attack criteria. For the 18 patients, 33 episodes of'hit by the wind' that met panic attack criteria were experienced in the previous month. For these 33 episodes, the most frequently reported DSM-IV panic attack symptoms were chills (100%; 33/33) and dizziness (88%; 29/33). Flashbacks played a role in the 'hit by the wind' episodes for 5 of the 18 patients (28%). In the discussion, a model of how the syndrome of 'hit by the wind' generates panic is adduced; also, possible Chinese origins of the disorder are discussed.

Audit of nasal steroid use and effectiveness in a rhinitis clinic.

BACKGROUND: Intranasal corticosteroids are the most effective treatment for allergic rhinitis based on meta-analyses of clinical trials; however, the population in such trials is restricted and may not be representative of patients who are later treated with the medication. The effectiveness of intranasal steroids in the real world has not been assessed. AIMS AND METHODS: We asked patients returning to a rhinitis clinic having been treated with intranasal corticosteroids for 3 months to fill in an anonymous questionnaire about their use of this treatment, its effectiveness and any adverse events. RESULTS: A total of 126 out of 134 patients completed the questionnaire. At least 119 of these had used the intranasal steroid spray; 79% found the sprays helpful, 40% found nasal blockage was reduced and 24% noted reduction of all symptoms. A total of 20% experienced no benefit, 21% noted minor adverse events - the most common being epistaxis in 9%. CONCLUSION: The real world effectiveness and adverse event profile of intranasal steroid sprays is similar in clinical practice to that seen in trials. The number needed to treat to obtain symptomatic benefit is 1.26 and to reduce all symptoms is 4. The number needed to harm is 11. Intranasal steroids are an effective and safe first line treatment for rhinitis.

Inflammatory focal myositis of the sternomastoid muscle: is there an absolute indication for biopsy? A case report and review of the literature.

Focal myositis is a localised inflammatory process affecting skeletal muscles belonging to the pathological group of inflammatory pseudo tumours of soft tissue that includes myositis ossificans, proliferative myositis and nodular pseudosarcomatous fasciitis. Very rarely, it may affect one of the neck muscles and present as a neck lump, in which case both the clinical and pathological picture can mimic a sarcoma. We describe a case of focal myositis of the sternocleidomastoid muscle, present a review of this rare condition and debate the necessity of biopsy.

A rare case of nasal glioma in the sphenoid sinus of an adult presenting with meningoencephalitis.

Nasal gliomas are rare embryologic development defects that commonly present shortly after birth. Very few cases have ever been reported in adults. Of those presenting in adult life, most were casual findings in patients with no symptoms. Rare localizations have been described in the nasopharynx, soft palate, orbital cave and scalp. We present an unusual case of a 56-year-old man who initially presented with meningoencephalitis and was found to have nasal glioma in the sphenoid sinus. Having discussed their embryology and histology, we conclude that MRI scanning is the preferred method of investigation. However, radiological evaluation preoperatively should not lead to any sense of security if no bony defect (and thus, the possibility of intracranial extension) is demonstrated. Extirpation via a functional surgical approach is the treatment of choice to prevent deformities in the facial bones. This can range from direct incision over the gliomata to removal transnasally by endoscopy or using a lateral rhinotomy approach depending on the location, size and involvement of the glioma.