Molecular phylogeny of elasmobranchs inferred from mitochondrial and nuclear markers

The elasmobranchs (sharks, rays and skates) being the extant survivors of one of the earliest offshoots of the vertebrate evolutionary tree are good model organisms to study the primitive vertebrate conditions. They play a significant role in maintaining the ecological balance and have high economic value. Due to over-exploitation and illegal fishing worldwide, the elasmobranch stocks are being decimated at an alarming rate. Appropriate management measures are necessary for restoring depleted elasmobranch stocks. One approach for restoring stocks is implementation of conservation measures and these measures can be formulated effectively by knowing the evolutionary relationship among the elasmobranchs. In this study, a total of 30 species were chosen for molecular phylogeny studies using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA gene and nuclear Internal Transcribed Spacer 2. Among different genes, the combined dataset of COI and 12S rRNA resulted in a well resolved tree topology with significant bootstrap/posterior probabilities values. The results supported the reciprocal monophyly of sharks and batoids. Within Galeomorphii, Heterodontiformes (bullhead sharks) formed as a sister group to Lamniformes (mackerel sharks): Orectolobiformes (carpet sharks) and to Carcharhiniformes (ground sharks). Within batoids, the Myliobatiformes formed a monophyly group while Pristiformes (sawfishes) and Rhinobatiformes (guitar fishes) formed a sister group to all other batoids.     

Preparation of Efficient Excision Repair Competent Cell-Free Extracts from C. reinhardtii Cells

Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers) in the DNA. We observed that excision repair (ER) synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC) DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.     

Comparing Accuracy of Wrist Intra-articular Needle Placement Via Ulnocarpal Approach by Training Level: A Cadaveric Study

BackgroundIntra-articular injections are a standard therapy and diagnostic tool for a variety of wrist conditions. Accurate needle placement is crucial for proper therapeutic benefit and prevention of complications. While some studies claim accurate needle placement requires imaging, others conclude that anatomical guidance is sufficient. This study aimed to evaluate the accuracy of intra-articular wrist needle placement with the ulnocarpal approach across differing levels of training using clinical anatomy alone.MethodsFourteen fresh-frozen, above-elbow cadaveric specimens were used. Intra-articular needle placement into the wrist via an ulnocarpal approach was attempted by nine study participants: two interns, two junior-level residents, two senior-level residents, two hand fellows, and one attending hand surgeon. Each injection was performed based on clinical examination and landmarks alone. The number of attempts and total time taken for each injection was recorded.ResultsOverall success rate was 71%, (89 of 126 attempts) and did not vary significantly across levels of training. Average time for needle placement among all participants was 10.9 ± 6.5 seconds. Timing of successful intra-articular needle placement (10.4 ± 5.2 seconds) significantly differed between levels. However, timing did not trend in any direction with more or less training. No significant difference was noted in total attempts or attempts with successful outcomes when comparing level of training.ConclusionThe ulnocarpal approach is a viable option for injection or aspiration of the wrist without image guidance. We were unable to show any relevant trends with timing or number of attempts in comparison to level of training. Level of Evidence: V     

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.     

Natural host-range and experimental transmission of Laem-Singh virus (LSNV)

Slow growth caused by viral diseases has become a major constraint in shrimp aquaculture. Laem-Singh virus (LSNV), a positive-sense single-stranded RNA (ssRNA) virus, has been identified in Penaeus monodon showing slow growth syndrome. To examine the host-range and transmission modes of the virus, 6 species of penaeid shrimp of varying life stages, sourced from the wild and from farms, as well as juvenile mud crabs Scylla serrata, were screened using RT-nested PCR. LSNV was detected in P. monodon, Fenneropenaeus merguiensis, Metapenaeus dobsoni, and Litopenaeus vannamei, but not in E indicus, Marsupenaeus japonicus or S. serrata. LSNV was most prevalent in P. monodon followed by M. dobsoni, F. merguiensis, and L. vannamei, and real-time quantitative RT-PCR (qRT-PCR) showed that LSNV infection loads were highest in P. monodon, followed by L. vannamei, M. dobsoni, and E merguiensis. The nucleotide sequence of the LSNV RdRP gene fragment amplified by RT-nested PCR was highly conserved (99% identity) across these 4 penaeid species. LSNV was detected in both small and normal-sized P. monodon collected from the same pond. In experimental infections of both P. monodon and S. serrata, LSNV infection loads increased over time. The present study extends the known natural penaeid host-range and geographical distribution of LSNV and shows for the first time the potential susceptibility of S. serrata.     

Antimony and bismuth dithiocarboxylates: Synthesis, characterization, crystal structures of [Sb(S2Ctol)3] and [Bi(S2CPh)3] and their transformation to polycrystalline M2S3 (M = Sb, Bi)

Antimony and bismuth dithiocarboxylates of the type [M(S₂CAr)₃] (M = Sb, Bi; Ar = C₆H₅ (Ph), 4-MeC₆H₄ (tol)) have been synthesized. These complexes were characterized by elemental analysis, IR, UV-Vis, and ¹H NMR spectroscopy. X-ray crystal structural analyses of [Sb(S₂Ctol)₃] and [Bi(S₂CPh)₃] showed that the dithiocarboxylates are asymmetrically chelated to metal atom. The latter acquires a distorted pentagonal pyramidal geometry as a consequence of the presence of a stereochemically active lone pair of electrons. Pyrolysis of these complexes either in a furnace or in refluxing diphenylether gave M₂S₃ as characterized by their XRD pattern and EDAX analysis.     

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than `mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.