Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection

Introduction: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. Aims: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. Methodology: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). Results: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. Conclusions: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK

Understanding prehospital blood transfusion decision-making for injured patients : an interview study

Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.Peer reviewe

Intratesticular Abscess in a Solitary Testicle: The Case for Testicle Sparing Management

We present the case of a 24-year-old immunocompromised man with an intratesticular abscess. The patient presented with one week of left scrotal pain and swelling. Workup included scrotal ultrasonography which revealed a large fluid collection within the tunica albuginea of the left testis. Surgical exploration of the left testis evacuated a significant amount of purulent fluid. The residual viable testicular parenchyma was salvaged. Intraoperative cultures grew Morganella morganii. Follow-up ultrasonography showed resolution of the testicular fluid collection, and the patient was discharged home with oral antibiotics. Intratesticular abscesses are a rare clinical entity which often result in orchiectomy

Synthesis, Purification and Crystallization of Guanine-rich RNA Oligonucleotides

Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution

miRGator: an integrated system for functional annotation of microRNAs

MicroRNAs (miRNAs) constitute an important class of regulators that are involved in various cellular and disease processes. However, the functional significance of each miRNA is mostly unknown due to the difficulty in identifying target genes and the lack of genome-wide expression data combining miRNAs, mRNAs and proteins. We introduce a novel database, miRGator, that integrates the target prediction, functional analysis, gene expression data and genome annotation. MiRNA function is inferred from the list of target genes predicted by miRanda, PicTar and TargetScanS programs. Statistical enrichment test of target genes in each term is performed for gene ontology, pathway and disease annotations. Associated terms may provide valuable insights for the function of each miRNA. For the expression analysis, miRGator integrates public expression data of miRNA with those of mRNA and protein. Expression correlation between miRNA and target mRNA/proteins is evaluated and their expression patterns can be readily compared. Our web implementation supports diverse query types including miRNA name, gene symbol, gene ontology, pathway and disease terms. Interfaces for exploring common targets or regulatory miRNAs and for profiling compendium expression data have been developed as well. Currently, miRGator, available at: http://genome.ewha.ac.kr/miRGator/, supports the human and mouse genomes

A novel class of microRNA-recognition elements that function only within open reading frames.

MicroRNAs (miRNAs) are well known to target 3' untranslated regions (3' UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these 'unusual' MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3' side rather than the 5' seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3' UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells

Suppression of Implanted MDA-MB 231 Human Breast Cancer Growth in Nude Mice by Dietary Walnut

Walnuts contain components that may slow cancer growth including omega 3 fatty acids, phytosterols, polyphenols, carotenoids, and melatonin. A pilot study was performed to determine whether consumption of walnuts could affect growth of MDA-MB 231 human breast cancers implanted into nude mice. Tumor cells were injected into nude mice that were consuming an AIN-76A diet slightly modified to contain 10% corn oil. After the tumors reached 3 to 5 mm diameter, the diet of one group of mice was changed to include ground walnuts, equivalent to 56 g (2 oz) per day in humans. The tumor growth rate from Day 10, when tumor sizes began to diverge, until the end of the study of the group that consumed walnuts (2.9 ± 1.1 mm3/day; mean ± standard error of the mean) was significantly less (P \u3e 0.05, t-test of the growth rates) than that of the group that did not consume walnuts (14.6 ± 1.3 mm 3 /day). The eicosapentaenoic and docosahexaenoic acid fractions of the livers of the group that consumed walnuts were significantly higher than that of the group that did not consume walnuts. Tumor cell proliferation was decreased, but apoptosis was not altered due to walnut consumption. Further work is merited to investigate applications to cancer in humans

Multicentre, prospective, open study to evaluate the safety and efficacy of hylan G-F 20 in knee osteoarthritis subjects presenting with pain following arthroscopic meniscectomy

The aim of the study was to evaluate the safety and efficacy of viscosupplementation with hylan G-F 20 in patients with mild to moderate osteoarthritis (OA) presenting with persistent knee pain 4–12 weeks after arthroscopic meniscectomy. A prospective, multi-centre, open study was carried out in patients with pain due to OA of the knee, not resolved by simple analgesics, 4–12 weeks after undergoing arthroscopic meniscectomy. To be eligible, patients had to score ≥50 mm and ≤90 mm on both walking pain and patient global assessment visual analogue scales (VAS; 0–100 mm) at baseline and be radiologically diagnosed pre-operatively with OA grade I or II on the Kellgren-Lawrence scale, with <50% joint space narrowing. Patients received three intra-articular, 2 ml injections of hylan G-F 20 in the target knee with an interval of 1 week between injections, and were followed for 52 weeks. The primary efficacy endpoint was the change from baseline in the walking pain VAS score at 26 weeks. Secondary outcome measures were the walking pain VAS scores at all other time points, the WOMAC Index at all time points, and patient and physician global assessment at all time points. The safety of the treatment was assessed using adverse event (AE) reports. A total of 62 patients (mean age 55.4 years, 52% male) were enrolled. The mean walking pain VAS score decreased by 36.8 mm from baseline at 26 weeks (P < 0.0001), and also showed statistically significant decreases (P < 0.0001) at all other time points. The change in WOMAC total and subscale scores from baseline were statistically significant (P < 0.0001) at all time points, as were the decreases in the physician and patient global assessment VAS scores. There were 18 target knee AEs (mostly pain and/or swelling and/or effusion) in 12 patients (19%) considered to be at least possibly related to treatment. The majority of these (78%) were mild or moderate in intensity. One patient (1.6%) experienced a serious adverse event (synovitis) in the target knee that was considered possibly related to study treatment. Hylan G-F 20 provides effective pain relief and improves stiffness and physical function in patients with mild to moderate OA presenting with persistent osteoarthritic pain 4–12 weeks after arthroscopic meniscectomy. Symptomatic efficacy was maximised at 12 weeks and maintained at 26 and 52 weeks. The type (pain and/or swelling and/or effusion) and the intensity (mostly mild/moderate) of AEs reported in this study are similar to those reported in other trials in different patient populations, but the incidence was higher (19%). The risk/benefit of hylan G-F 20 in this particular population of patients is favourable

Flowering response of pearl millet to water stress during panicle development.

In pearl millet, severe water deficit during the period of panicle development delays flowering. The flowering response of both main shoot and tillers to water stress during panicle development was investigated using four hybrids. Panicle initiation of all tillers occurred in the three early genotypes despite water stress. In the late genotype, however, panicle initiation of tillers occurred only after the release of stress. The delay in flowering due to water stress was more pronounced in the tillers than in the main shoot. However, the proportion of tillers producing an inflorescence was increased by water stress. Grain yield losses on the main shoot by water stress were compensated by an increase in tiller grain yields. Delay in flowering and buffering by tillers provide an important adaptive mechanism to overcome a period of drought stress prior to flowering