poly(UG)-tailed RNAs in genome protection and epigenetic inheritance.

Mobile genetic elements threaten genome integrity in all organisms. RDE-3 (also known as MUT-2) is a ribonucleotidyltransferase that is required for transposon silencing and RNA interference in Caenorhabditis elegans1-4. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (designated poly(UG) or pUG tails)5. Here we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to targets of RNA interference, as well as to transposon RNAs. RNA fragments attached to pUG tails with more than 16 perfectly alternating 3' U and G nucleotides become gene-silencing agents. pUG tails promote gene silencing by recruiting RNA-dependent RNA polymerases, which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed pUG RNA biogenesis underlie double-stranded-RNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA-siRNA silencing loop enables parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.

Spatiotemporal regulation of liquid-like condensates in epigenetic inheritance.

Non-membrane-bound organelles such as nucleoli, processing bodies, Cajal bodies and germ granules form by the spontaneous self-assembly of specific proteins and RNAs. How these biomolecular condensates form and interact is poorly understood. Here we identify two proteins, ZNFX-1 and WAGO-4, that localize to Caenorhabditis elegans germ granules (P granules) in early germline blastomeres. Later in germline development, ZNFX-1 and WAGO-4 separate from P granules to define an independent liquid-like condensate that we term the Z granule. In adult germ cells, Z granules assemble into ordered tri-condensate assemblages with P granules and Mutator foci, which we term PZM granules. Finally, we show that one biological function of ZNFX-1 and WAGO-4 is to interact with silencing RNAs in the C. elegans germline to direct transgenerational epigenetic inheritance. We speculate that the temporal and spatial ordering of liquid droplet organelles may help cells to organize and coordinate the complex RNA processing pathways that underlie gene-regulatory systems, such as RNA-directed transgenerational epigenetic inheritance.

Acquisition of new protein domains by coronaviruses: analysis of overlapping genes coding for proteins N and 9b in SARS coronavirus.

Acquisition of new proteins by viruses usually occurs through horizontal gene transfer or through gene duplication, but another, less common mechanism is the usage of completely or partially overlapping reading frames. A case of acquisition of a completely new protein through introduction of a start codon in an alternative reading frame is the protein encoded by open reading frame (orf) 9b of SARS coronavirus. This gene completely overlaps with the nucleocapsid (N) gene (orf9a). Our findings indicate that the orf9b gene features a discordant codon-usage pattern. We analyzed the evolution of orf9b in concert with orf9a using sequence data of betacoronavirus-lineage b and found that orf9b, which encodes the overprinting protein, evolved largely independent of the overprinted orf9a. We also examined the protein products of these genomic sequences for their structural flexibility and found that it is not necessary for a newly acquired, overlapping protein product to be intrinsically disordered, in contrast to earlier suggestions. Our findings contribute to characterizing sequence properties of newly acquired genes making use of overlapping reading frames.

Recurrent rhinosporidiosis in a young male: A case report and its taxonomical journey.

Rhinosporidiosis is a rarely encountered granulomatous infection caused by Rhinosporidium seeberi affecting both humans and animals. Although the disease has been reported worldwide, it is mainly endemic in tropical and subtropical countries. In the Indian subcontinent, it is endemic in some parts like Orissa, Tamil Nadu, Kerala, eastern Madhya Pradesh, and Chhattisgarh. It is a chronic granulomatous disease with varied controversial taxonomical history, but recently based on genetic sequencing and the nature of aquatics, it was later identified as an aquatic eukaryote. The mucous membranes are frequently impacted in humans, with a typical manifestation being the presence of a polypoidal mass. The occurrence of Rhinosporidiosis in nonendemic regions is uncommon. We report one such case of a young male with recurrent Rhinosporidiosis from India.

Gustatory receptor neurons in Manduca sexta contain a TrpA1-dependent signaling pathway that integrates taste and temperature.

Temperature modulates the peripheral taste response of many animals, in part by activating transient receptor potential (Trp) cation channels. We hypothesized that temperature would also modulate peripheral taste responses in larval Manduca sexta. We recorded excitatory responses of the lateral and medial styloconic sensilla to chemical stimuli at 14, 22, and 30 °C. The excitatory responses to 5 chemical stimuli-a salt (KCl), 3 sugars (sucrose, glucose, and inositol) and an alkaloid (caffeine)-were unaffected by temperature. In contrast, the excitatory response to the aversive compound, aristolochic acid (AA), increased robustly with temperature. Next, we asked whether TrpA1 mediates the thermally dependent taste response to AA. To this end, we 1) identified a TrpA1 gene in M. sexta; 2) demonstrated expression of TrpA1 in the lateral and medial styloconic sensilla; 3) determined that 2 TrpA1 antagonists (HC-030031 and mecamylamine) inhibit the taste response to AA, but not caffeine; and then 4) established that the thermal dependence of the taste response to AA is blocked by HC-030031. Taken together, our results indicate that TrpA1 serves as a molecular integrator of taste and temperature in M. sexta.

Identification of chemosensory receptor genes in Manduca sexta and knockdown by RNA interference.

BACKGROUND: Insects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. Although our understanding of the molecular genetic basis for Drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. The tobacco hornworm, Manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. It is also a major agricultural pest on solanaceous crops. However, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. The ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in M. sexta would be greatly beneficial. RESULTS: Here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. We also used dsRNA feeding as a method to induce RNA interference in larval chemosensory tissues. CONCLUSIONS: We report identification of new chemosensory receptor genes including 17 novel odorant receptors and one novel gustatory receptor. Further, we demonstrate that systemic RNA interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation.

An atypical case of giant intradiploic epidermoid tumor.

Intradiploic epidermoid tumors are uncommon and giant epidermoid with dural involvement is scarcer. We report a unique case of a giant frontal epidermoid tumor presenting without typical features of swelling or bulge in scalp. A 61-year-old male presented with the complaints of forgetfulness and headache. Contrast magnetic resonance imaging brain revealed a large left frontal epidermoid tumor. A tumor measuring 13 × 11 × 4 cm, involving the dura but sparing the brain parenchyma, was excised through left frontal craniotomy. Such a presentation of giant epidermoid tumor with dural involvement is highly unusual. Complete surgical excision is the final aim and vigilant follow-up for recurrence is a must.

piRNAs coordinate poly(UG) tailing to prevent aberrant and perpetual gene silencing.

Noncoding RNAs have emerged as mediators of transgenerational epigenetic inheritance (TEI) in a number of organisms. A robust example of such RNA-directed TEI is the inheritance of gene-silencing states following RNA interference (RNAi) in the metazoan C. elegans. During RNAi inheritance, gene silencing is transmitted by a self-perpetuating cascade of siRNA-directed poly(UG) tailing of mRNA fragments (pUGylation), followed by siRNA synthesis from poly(UG)-tailed mRNA templates (termed pUG RNA/siRNA cycling). Despite the self-perpetuating nature of pUG RNA/siRNA cycling, RNAi inheritance is finite, suggesting that systems likely exist to prevent indefinite RNAi-triggered gene silencing. Here we show that, in the absence of Piwi-interacting RNAs (piRNAs), an animal-specific class of small noncoding RNA, RNAi-based gene silencing can become essentially permanent, lasting at near 100% penetrance for more than 5 years and hundreds of generations. This perpetual gene silencing is mediated by continuous pUG RNA/siRNA cycling. Further, we find that piRNAs coordinate endogenous RNAi pathways to prevent germline-expressed genes, which are not normally subjected to TEI, from entering a state of continual and irreversible epigenetic silencing also mediated by continuous maintenance of pUG RNA/siRNA cycling. Together, our results show that one function of C. elegans piRNAs is to insulate germline-expressed genes from aberrant and runaway inactivation by the pUG RNA/siRNA epigenetic inheritance system.

Rhino-orbital-cerebral mucormycosis in times of COVID-19: A neurosurgical experience.

BACKGROUND: The gravity of "second wave" of COVID-19 has effaced many new challenges in India; mucormycosis being a recent one. Diabetes mellitus (DM) is a known significant risk factor for mucormycosis. Here, we present our experience with rhino-orbital-cerebral mucormycosis (ROCM) during the "second wave of COVID-19" at a tertiary health care centre in North India. METHODS: This case series includes four cases of ROCM that were managed by our neurosurgical team in view ofcerebral involvement. RESULTS: All the cases with an exception of one (Case 1), had a history of treatment for COVID-19 pneumonia. Case 2, 3 had undergone functional endoscopic sinus surgery (FESS) and orbital decompression before the onset of cerebral involvement; Case 4 underwent FESS and cranial surgery in the same sitting. All the patients had a history of DM and all the cases treated for COVID-19 pneumonia had a history of treatment with corticosteroids. Two patients underwent surgery with the exception of one patient, who did not provide consent for the same. One patient expired before surgical excision could be attempted. CONCLUSION: Regular and intensive follow-up is the key in swift detection and management of ROCM in post-COVID patients. While surgical excision is advisable in the fungal lesion, it must be borne in mind that radical excision of cerebral lesions is associated with morbidity, delayed recovery, and prolonged ICU stay. Culture and sensitivity-based antibiotics should be used judiciously as fever is a common postoperative complication. Blood sugar monitoring and control of DM are paramount in this condition. Steroids should be avoided in the management of cerebral edema with judicious use of hypertonic saline or mannitol.

The RNAi Inheritance Machinery of Caenorhabditis elegans.

Gene silencing mediated by dsRNA (RNAi) can persist for multiple generations in Caenorhabditis elegans (termed RNAi inheritance). Here we describe the results of a forward genetic screen in C. elegans that has identified six factors required for RNAi inheritance: GLH-1/VASA, PUP-1/CDE-1, MORC-1, SET-32, and two novel nematode-specific factors that we term here (heritable RNAi defective) HRDE-2 and HRDE-4 The new RNAi inheritance factors exhibit mortal germline (Mrt) phenotypes, which we show is likely caused by epigenetic deregulation in germ cells. We also show that HRDE-2 contributes to RNAi inheritance by facilitating the binding of small RNAs to the inheritance Argonaute (Ago) HRDE-1 Together, our results identify additional components of the RNAi inheritance machinery whose conservation provides insights into the molecular mechanism of RNAi inheritance, further our understanding of how the RNAi inheritance machinery promotes germline immortality, and show that HRDE-2 couples the inheritance Ago HRDE-1 with the small RNAs it needs to direct RNAi inheritance and germline immortality.

A reference gene set for chemosensory receptor genes of Manduca sexta.

The order of Lepidoptera has historically been crucial for chemosensory research, with many important advances coming from the analysis of species like Bombyx mori or the tobacco hornworm, Manduca sexta. Specifically M. sexta has long been a major model species in the field, especially regarding the importance of olfaction in an ecological context, mainly the interaction with its host plants. In recent years transcriptomic data has led to the discovery of members of all major chemosensory receptor families in the species, but the data was fragmentary and incomplete. Here we present the analysis of the newly available high-quality genome data for the species, supplemented by additional transcriptome data to generate a high quality reference gene set for the three major chemosensory receptor gene families, the gustatory (GR), olfactory (OR) and antennal ionotropic receptors (IR). Coupled with gene expression analysis our approach allows association of specific receptor types and behaviors, like pheromone and host detection. The dataset will provide valuable support for future analysis of these essential chemosensory modalities in this species and in Lepidoptera in general.

Accessory proteins of SARS-CoV and other coronaviruses.

The huge RNA genome of SARS coronavirus comprises a number of open reading frames that code for a total of eight accessory proteins. Although none of these are essential for virus replication, some appear to have a role in virus pathogenesis. Notably, some SARS-CoV accessory proteins have been shown to modulate the interferon signaling pathways and the production of pro-inflammatory cytokines. The structural information on these proteins is also limited, with only two (p7a and p9b) having their structures determined by X-ray crystallography. This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins, with an emphasis on their involvement in virus-host interaction. The accessory proteins of other coronaviruses are also briefly discussed. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses" (see Introduction by Hilgenfeld and Peiris (2013)).