Isolation of a putative probiotic strain S12 and its effect on growth performance, non-specific immunity and disease-resistance of white shrimp, Litopenaeus vannamei.

The common pathogens in aquaculture are very different from those in terrestrial animals. The objective of this study was to isolate probiotic strain (s) from the digestive tract of healthy white shrimp Litopenaeus vannamei which was effective against aquatic animal pathogens. The putative probiotic strain S12 was identified as Bacillus subtilis based on the morphological and biochemical properties and 16S rDNA gene sequencing. The L. vannamei were fed with five different diets: control (basal diet with no probiotics or antibiotics), antibiotic control (basal diet supplemented with 0.3% florfenicol), basal diet supplemented with 5 × 10(9) cfu kg(-1) , 5 × 10(10) cfu kg(-1) and 5 × 10(11) cfu kg(-1) probiotic S12 (PS1-3). Each diet was randomly fed to quadruplication groups of 40 shrimps (0.4 ± 0.01 g) reared in tanks. After an 8-week feeding, the survival rate of shrimps fed with PS1 and PS3 were the highest among all treatments (P < 0.05). The moisture content of shrimps fed with florfenicol was significantly lower than that of the control group (P < 0.05). The supplement of probiotic S12 decreased the body crude lipid significantly (P < 0.05). The activities of phagocytic rate, lysozyme (LZ), superoxide dismutase phenoloxidase (SOD) and antibacterial activity were significantly higher than those in the control (P < 0.05), and the activities of SOD and the antibacterial activity in PS2 and PS3 were significantly higher than those in antibiotic control (P < 0.05). When infected with Vibrio harveyi at 4-weeks, the mortality was significantly lower (P < 0.05) in PS2 and PS3 groups than that in the control. After being infected with V. harveyi at 8-weeks, the mortality was significantly lower in the probiotic and antibiotic groups than that in the control (P < 0.05). This study suggested that probiotics could be used as an effective immunopotentiator, the optimal dose of the probiotic strain S12 is 5 × 10(10) cfu kg(-1) diet.

Beneficial effects of the first case of washed microbiota transplantation for postorgasmic illness syndrome: a case report.

Introduction: Postorgasmic illness syndrome (POIS) is characterized by allergic symptoms and flu-like illness after ejaculation. There are still no effective treatments for POIS. Aim: To report the first case of washed microbiota transplantation (WMT) to treat patient with POIS. Methods: Data were collected from a patient with POIS who had received 3 courses of WMT: self-rating scale of POIS symptoms, Self-rating Anxiety Scale, Self-rating Depression Scale, and Symptom Checklist 90. The patient's stool samples for 16sDNA sequencing were collected 1 month after WMT. Results: POIS symptoms improved after WMT. Scores decreased from baseline after WMT: self-rating scale of POIS symptoms (before WMT, 16; after first, 16; after second, 8; after third, 9), Self-rating Anxiety Scale (45, 42.5, 37.5, 45), Self-rating Depression Scale (63.75, 58.75, 47.5, 50), and Symptom Checklist 90 (143, 140, 109, 149). Characteristics of the patient's gut microbiota changed. At the genus level, the relative abundance of beneficial bacteria increased, and some opportunistic pathogenic bacteria decreased. Conclusion: WMT may be an effective and safe choice for the treatment of patients with POIS by changing the gut microbiota of the host.

Multiprotease improves amino acid release in vitro, energy, and nutrient utilization in broilers fed diets varying in crude protein levels.

Low crude protein (CP) diets can reduce nitrogen (N) excretion and costs by increasing N utilization efficiency. Exogenous proteases may further improve protein digestibility in low CP diets. This study first evaluated in vitro the efficacy of a multiprotease on amino acid (AA) release from feedstuffs and broiler feed. Later, a broiler study evaluated the effect of feeding corn-soybean meal diets containing 3 CP levels (17, 19, and 21% CP) with supplementation on top of 0 or 2,400 U/kg multiprotease on chicken growth performance, total tract CP, and ileal AA digestibilities, and energy utilization. Ross 708 male chickens were placed in 42 cages and assigned to 6 treatments resulting from a 3 × 2 factorial arrangement. Three isocaloric basal diets were formulated to reduce CP, but all diets maintained digestible Lys:CP in 5.47% and the same ideal protein profile. Data were analyzed in a completely randomized design. On average, the multiprotease increased (P < 0.05) in vitro free AA release by 27.81% in most feedstuffs evaluated compared to the control. For broiler feed, 1,200 U/g multiprotease addition improved (P < 0.001) in vitro free AA release by 18.90%. This multiprotease showed interaction effects (P < 0.05) on chicken FCR, energy, and CP digestibility. As expected, BW at 24 d, BW gain, and FCR (8-24 d) worsened (P < 0.001) as dietary CP reduced from 21 to 17%, and multiprotease addition did not improve (P > 0.05) these parameters. BW gain decreased by 12.9% when N intake was reduced from 49.32 to 38.49 g/bird. Multiprotease supplementation improved (P < 0.01) AMEn by 71 kcal/kg, CP digestibility from 59.45 to 63.51%, ileal AA digestibility, and DM digestibility from 67.08 to 73.49%, but only in the 21% CP diet. No differences in ileal AA digestibility due to CP level (P > 0.05) were detected, except for Cys digestibility (P < 0.01). In conclusion, low CP diets reduced growth performance and improved N utilization but negatively affected energy utilization efficiency. Exogenous multiprotease supplementation improved AME, AMEn, protein, ileal AA, and DM digestibility in the 21% CP diet without significantly affecting growth performance.

Mitogenome of a monotypic genus, Oliotius Kottelat, 2013 (Cypriniformes: Cyprinidae): Genomic characterization and phylogenetic position.

We examined the evolutionary status of the genus Oliotius Kottelat, 2013, clarified the mitogenome structure of Smiliogastrinae, and analyzed its phylogenetic information to provide a reference for the taxonomy, evolutionary genetics, and germplasm identification of Smiliogastrinae. Using next-generation sequencing, the complete mitogenome sequence of the monotypic species Oliotius oligolepis was obtained, annotated, and characterized. The mitogenomic sequences of Smiliogastrinae fish were compared and analyzed, and a phylogenetic tree was constructed. The mitogenome of O. oligolepis was 16,636 base pairs long and contained 22 tRNAs, 13 protein-coding genes, 2 rRNAs, and 1 D-loop. The Smiliogastrinae species exhibited gene arrangement consistency, with slight differences in their genome lengths and compositions. Among the 13 protein-coding genes, ND3 showed the highest polymorphism rate and largest genetic distance. Our phylogenetic analysis showed that the target species O. oligolepis is closely related to Barbodes binotatus. The genus Oliotius is monotypic, and the genus Barbodes is not supported as a monophyletic group. This molecular information provides a foundation for further research on the phylogeny of the genera Oliotius and Barbodes and provides insight into the phylogenetic features of fishes belonging to Smiliogastrinae.

DNA barcoding of Scomberomorus (Scombridae, Actinopterygii) reveals cryptic diversity and misidentifications.

The genus Scomberomorus is economically important; however, the taxonomic status and phylogenetic relationships in this genus are not clearly resolved, making it difficult to effectively protect and exploit fish resources. To clarify the taxonomic status of Scomberomorus species, mitochondrial cytochrome c oxidase I (COI) gene sequences of 150 samples were analyzed. The average genetic distance among 14 species was approximately 11 times greater than the distances within species, in accordance with the '10× rule' of species identification. Five of the 14 species did not form monophyletic clades based on a Bayesian inference gene tree. The application of four DNA-based species delimitation methods (automatic barcode gap discovery, barcode index numbers, Poisson tree process, and the K/θ method) yielded several key results. (1) Cryptic species were detected within Scomberomoruscommerson. (2) A Scomberomorusqueenslandicus sample from Australia was misidentified as S.commerson in the Barcode of Life Data System (BOLD). (3) Specimens originally identified as Scomberomorusguttatus was differentiated into four OTUs or species, two in the Yellow, South China, and Java seas, and two in geographically distant areas, one each in the Arabian Sea and the Bay of Bengal. (4) Six specimens from South Africa originally identified as S.plurilineatus most likely do not belong to the species. (5) Specimens identified as S.maculatus and S.regalis were conspecific; however, introgression cannot be ruled out. Our findings revealed cryptic diversity and difficulties in morphological identification of species in the genus Scomberomorus. This study provides scientifically based support for the conservation of germplasm resources of the genus Scomberomorus.

Synaptotagmin-1 and -7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells.

Synaptotagmin-1, the canonical isoform of the synaptotagmin family, is a Ca(2+) sensor for fast synchronous neurotransmitter release in forebrain neurons and chromaffin cells. Even though deletion of synaptotagmin-1 abolishes fast exocytosis in chromaffin cells, it reduces overall secretion by only 20% because of the persistence of slow exocytosis. Therefore, another Ca(2+) sensor dominates release in these cells. Synaptotagmin-7 has a higher Ca(2+) affinity and slower binding kinetics than synaptotagmin-1, matching the proposed properties for the second, slower Ca(2+) sensor. Here, we examined Ca(2+)-triggered exocytosis in chromaffin cells from KO mice lacking synaptotagmin-7, and from knockin mice containing normal levels of a mutant synaptotagmin-7 whose C(2)B domain does not bind Ca(2+). In both types of mutant chromaffin cells, Ca(2+)-triggered exocytosis was decreased dramatically. Moreover, in chromaffin cells lacking both synaptotagmin-1 and -7, only a very slow release component, accounting for approximately 30% of WT exocytosis, persisted. These data establish synaptotagmin-7 as a major Ca(2+) sensor for exocytosis in chromaffin cells, which, together with synaptotagmin-1, mediates almost all of the Ca(2+) triggering of exocytosis in these cells, a surprising result, considering the lack of a role of synaptotagmin-7 in synaptic vesicle exocytosis.

Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis.

Synaptotagmin-7 is a candidate Ca(2+) sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca(2+) sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C(2)B domains that exhibit Ca(2+)-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca(2+) sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca(2+)-binding properties. Here, we examine how synaptotagmin-7 binds Ca(2+) and test whether this Ca(2+) binding regulates Ca(2+)-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca(2+)-binding mode similar to that of the synaptotagmin-1 C(2)A domain, suggesting that the synaptotagmin-1 and -7 C(2) domains generally employ comparable Ca(2+)-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca(2+) binding either to both C(2) domains of synaptotagmin-7 or only to its C(2)B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca(2+) binding to both C(2) domains caused an approximately 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca(2+) binding to only the C(2)B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca(2+) binding to the synaptotagmin-7 C(2) domains is physiologically important for stabilizing synaptotagmin-7, but that Ca(2+) binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca(2+)-dependent synaptic exocytosis.

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice.

Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas.

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

N-glycosylation is essential for vesicular targeting of synaptotagmin 1.

Synaptotagmins 1 and 7 are candidate Ca(2+) sensors for exocytosis localized to synaptic vesicles and plasma membranes, respectively. We now show that the N-terminal intraluminal sequence of synaptotagmin 1, when transplanted onto synaptotagmin 7, redirects synaptotagmin 7 from the plasma membrane to secretory vesicles. Conversely, mutation of the N-terminal N-glycosylation site of synaptotagmin 1 redirects synaptotagmin 1 from vesicles to the plasma membrane. In cultured hippocampal neurons, the plasma membrane-localized mutant of synaptotagmin 1 suppressed the readily releasable pool of synaptic vesicles, whereas wild-type synaptotagmin 1 did not. In addition to the intraluminal N-glycosylation site, the cytoplasmic C(2) domains of synaptotagmin 1 were required for correct targeting but could be functionally replaced by the C(2) domains of synaptotagmin 7. Our data suggest that the intravesicular N-glycosylation site of synaptotagmin 1 collaborates with its cytoplasmic C(2) domains in directing synaptotagmin 1 to synaptic vesicles via a novel N-glycosylation-dependent mechanism.

Rhodium(III)-catalyzed C-H/C-C activation sequence: vinylcyclopropanes as versatile synthons in direct C-H allylation reactions.

Succession of C-H activation and C-C activation was achieved by using a single rhodium(III) catalyst. Vinylcyclopropanes were used as versatile coupling partners. Mechanistic studies suggest that the olefin insertion step is rate-determining and a facile ß-carbon elimination is involved, which represents a novel ring opening mode of vinylcyclopropanes.

The N-terminal domain of the large subunit of human replication protein A binds to Werner syndrome protein and stimulates helicase activity.

Werner syndrome (WS) is a recessive inherited human disease characterized by the early onset of aging. The gene mutated in WS encodes a DNA helicase that unwinds the double helical structure of DNA in the 3'-->5' direction as well as a 3'-->5' exonuclease. Our previous studies indicated that the activity of Werner syndrome helicase (WRN) could be stimulated by human replication protein A (hRPA), a heterotrimeric single-stranded DNA binding protein. We now localize the interaction between WRN and hRPA by measuring the stimulation of helicase activity and the binding of WRN by hRPA and its derivatives. The large subunit of hRPA (hRPA70) stimulates WRN helicase to the same extent as the hRPA heterotrimer, whereas the dimer of the two smaller subunits (hRPA 32.14) does not stimulate. By examining hRPA70 mutants with progressive deletions from either the C- or N-terminus, we found that the domain responsible for stimulation lies in the N-terminal half of the protein. By using enzyme-linked immunosorbent assay (ELISA) to examine physical interaction between WRN and the same deletion mutants, we found that the WRN-binding motif is located within amino acids 100-300 and overlaps with the single-stranded DNA binding domain (amino acids 150-450). We suggest that hRPA, by engaging in both protein-protein and protein-DNA interactions, facilitates unwinding events catalyzed by WRN helicase during DNA synthetic processes. These data should help further elucidation of the molecular mechanisms of genetic instability and premature aging phenotypes manifested by WS.

Mild rhodium(III)-catalyzed C-H allylation with 4-vinyl-1,3-dioxolan-2-ones: direct and stereoselective synthesis of (E)-allylic alcohols.

A rhodium(III)-catalyzed C-H direct allylation reaction with 4-vinyl-1,3-dioxolan-2-ones has been developed. The reaction provides a facile and stereoselective access to substituted-(E)-allylic alcohols under mild and redox-neutral reaction conditions. Olefinic C-H activation is applicable, giving multifunctionalized skipped dienes in good yields. Minimal double-bond migration was observed.

The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction.

Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities.

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.