Microscopic investigation and pharmacognostic evaluation of leaves of Anemone rupicola Camb.

There is almost no scientific literature available on the phytochemistry and pharmacognosy of this plant so the basic aim of the study is to analyze pharmacognostic features of dried leaves of Anemone rupicola Camb. Morphology was studied by using the features like margins, shape, length, width, surface and so forth. For anatomical examination leaves were preserved in formalin acetic alcohol (FAA) and cutting the thin section of leaves and petiole with razor blade. Macroscopic characters of powdered drug were evaluated through organoleptic method. Qualitative phytochemical analysis was done by adopting the standard procedures. The antimicrobial activity of methanolic extract of Anemone rupicola Camb. was performed by Agar well diffusion method. The powder microscopic study showed epidermal cells, trichomes, various type of tracheids, pitted vessels, and sclerenchymatous fibers. Transverse cutting of leaves and petiole revealed the presence of different cells such as epidermis, palisade parenchyma, spongy parenchyma cells, aerenchyma, collenchyma, trichomes, and vascular bundles. The investigation of phytochemicals in distilled water, methanol and in ethanol solvent indicated the presence of various secondary compounds such as phenols, saponins, glycosides, alkaloids, tannins, cardiac glycosides, terpenoids, coumarins, and flavonoids. The methanolic extract of Anemone rupicola Camb. has potential as an antimicrobial agent. However, further studies are needed to confirm its efficacy and safety. This study can be used as powerful tool for the identification and authentication of this plant. For the purity and quality control these results will be helpful tool. This will also shed light into new areas where researchers can intervene in developing new drugs for future use. RESEARCH HIGHLIGHTS: Morphological Features of the Anemone rupicola Anatomical examination of leaves of Anemeone rupicola Microscopic features of powdered drug of leaves of Anemone rupicola Qualitative Phytochemical analysis of the leaves of Anemone rupicola.

Pregnancy and calving rates improved using modified swim-up method for buffalo semen sexing.

Present study aimed to evaluate field fertility rate and calf sex ratio of Nili Ravi buffalo semen sexed through modified swim-up method (Animal Reproduction Science, 182, 2017, 69). For this purpose, five mature Nili-Ravi buffalo bulls kept at semen production unit, Qadirabad, Pakistan, were selected. Two consecutive ejaculates per week were collected with artificial vagina for 3 weeks. Qualified semen ejaculates were pooled and divided into two aliquots. The first aliquot was processed by routine procedure (control), whereas the second was processed by modified swim-up technique. After separation, semen was diluted in tris-citric acid extender and cryopreserved using standard techniques. Sexed semen was evaluated for fertility trials during peak breeding season. Artificially inseminated animals were examined for pregnancy rate through rectal palpation at least 3 months after insemination under field conditions. Calving ratio of female and male calves were recoded after Parturition. The fertility rate was higher (p < .05) in X-sorted sperm (70%) as compared with control (47%). The female calf ratio was higher (p < .05) in X-sorted sperm (78.58%) compared with control (53.3%). In Conclusion, conception rate and production of female calf were significantly higher with sexed semen separated through modified swim-up method compared with unsexed control.

Characterization of the Phase-Variable Autotransporter Lav Reveals a Role in Host Cell Adherence and Biofilm Formation in Nontypeable Haemophilus influenzae.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation. A survey of Haemophilus species genome sequences showed lav is present in ∼60% of NTHi strains, but lav is not present in most typeable H. influenzae strains. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.

Streptococcus suis Encodes Multiple Allelic Variants of a Phase-Variable Type III DNA Methyltransferase, ModS, That Control Distinct Phasevarions.

Streptococcus suis is a significant cause of bacterial meningitis in humans, particularly in Southeast Asia, and is a leading cause of respiratory and invasive disease in pigs. Phase-variable DNA methyltransferases, associated with restriction-modification (R-M) systems, are a source of epigenetic gene regulation, controlling the expression of multiple genes. These systems are known as phasevarions (phase-variable regulons) and have been characterized in many host-adapted bacterial pathogens. We recently described the presence of a Type III DNA methyltransferase in S. suis, ModS, which contains a simple sequence repeat (SSR) tract within the open reading frame of the modS gene and which differed in length between individual strains. We also observed that multiple allelic variants of the modS gene were present in a population of S. suis isolates. Here, we demonstrate that a biphasic ON-OFF switching of expression occurs in the two most common ModS alleles, ModS1 and ModS2, and that switching is dependent on SSR tract length. Furthermore, we show using single-molecule real-time (SMRT) sequencing that ModS1 and ModS2 are active methyltransferases in S. suis ON-OFF switching of each ModS allele results in the regulation of distinct phasevarions, with the ModS2 phasevarion impacting growth patterns and antibiotic resistance. This is the first demonstration of a phase-variable Type III DNA methyltransferase in a Gram-positive organism that controls a phasevarion. Characterizing the phenotypic effects of phasevarions in S. suis is key to understanding pathogenesis and the development of future vaccines.IMPORTANCEStreptococcus suis is a causative agent of meningitis, polyarthritis, and polyserositis in swine, and it is a major cause of zoonotic meningitis in humans. Here, we investigate epigenetic gene regulation in S. suis by multiple phasevarions controlled by the phase-variable Type III DNA methyltransferase ModS. This is the first characterized example of a Type III R-M system regulating a phasevarion in a Gram-positive organism. We demonstrate that biphasic ON-OFF switching of ModS expression results in differences in bacterial growth and antibiotic resistance. Understanding the effects of ModS phase variation is required to determine the stably expressed antigenic repertoire of S. suis, which will direct and inform the development of antimicrobial treatments and vaccines against this important pathogen.

Seasonality and photoperiod influence in vitro production of buffalo embryos.

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January-March, April-June, July-September and October-December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April-June) and maximum during fourth quarter (October-December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.

Sperm miR-15a and miR-29b are associated with bull fertility.

MicroRNAs modulate male fertility by regulating gene expression. In this study, dynamics of sperm miR-15a, miR-29b and miR-34a from high fertility (HF) and low fertility (LF) bulls using RT-qPCR were evaluated. Bioinformatic tools were employed to ascertain genes of interest of the sperm miRNAs. The expression levels of p53, BCL2, BAX and DNMT1 in bull spermatozoa were determined by immunoblotting. MicroRNA levels of miR-15a and miR-29 were higher in LF sires when compared with those present in HF bulls. Expression levels of miR-34a did not differ between the two groups. We found an inverse correlation between miR-15a and bull fertility. MiR29-b was also negatively associated with fertility scores. BCL2 and DNMT1 were higher in HF bulls while BAX was higher in the LF group. Our data showed a positive correlation between BCL2 and bull fertility. In addition, DNMT1 was positively associated with bull fertility. Furthermore, levels of BAX were negatively linked with bull fertility scores. Identification of miRNAs found in the spermatozoa of sires with different in vivo fertility helps understand the alterations in the fertilising capacity from cattle and other mammals. These potential biomarkers can be used in reproductive biotechnology as fertility markers to assess semen quality and predict male fertility.

Phasevarions of bacterial pathogens - phase-variable epigenetic regulators evolving from restriction-modification systems.

Phase-variable DNA methyltransferases control the expression of multiple genes via epigenetic mechanisms in a wide variety of bacterial species. These systems are called phasevarions, for phase-variable regulons. Phasevarions regulate genes involved in pathogenesis, host adaptation and antibiotic resistance. Many human-adapted bacterial pathogens contain phasevarions. These include leading causes of morbidity and mortality worldwide, such as non-typeable Haemophilus influenzae, Streptococcus pneumoniae and Neisseria spp. Phase-variable methyltransferases and phasevarions have also been discovered in environmental organisms and veterinary pathogens. The existence of many different examples suggests that phasevarions have evolved multiple times as a contingency strategy in the bacterial domain, controlling phenotypes that are important in adapting to environmental change. Many of the organisms that contain phasevarions have existing or emerging drug resistance. Vaccines may therefore represent the best and most cost-effective tool to prevent disease caused by these organisms. However, many phasevarions also control the expression of current and putative vaccine candidates; variable expression of antigens could lead to immune evasion, meaning that vaccines designed using these targets become ineffective. It is therefore essential to characterize phasevarions in order to determine an organism's stably expressed antigenic repertoire, and rationally design broadly effective vaccines.

Methionine biosynthesis and transport are functionally redundant for the growth and virulence of Salmonella Typhimurium.

Methionine (Met) is an amino acid essential for many important cellular and biosynthetic functions, including the initiation of protein synthesis and S-adenosylmethionine-mediated methylation of proteins, RNA, and DNA. The de novo biosynthetic pathway of Met is well conserved across prokaryotes but absent from vertebrates, making it a plausible antimicrobial target. Using a systematic approach, we examined the essentiality of de novo methionine biosynthesis in Salmonella enterica serovar Typhimurium, a bacterial pathogen causing significant gastrointestinal and systemic diseases in humans and agricultural animals. Our data demonstrate that Met biosynthesis is essential for S. Typhimurium to grow in synthetic medium and within cultured epithelial cells where Met is depleted in the environment. During systemic infection of mice, the virulence of S. Typhimurium was not affected when either de novo Met biosynthesis or high-affinity Met transport was disrupted alone, but combined disruption in both led to severe in vivo growth attenuation, demonstrating a functional redundancy between de novo biosynthesis and acquisition as a mechanism of sourcing Met to support growth and virulence for S. Typhimurium during infection. In addition, our LC-MS analysis revealed global changes in the metabolome of S. Typhimurium mutants lacking Met biosynthesis and also uncovered unexpected interactions between Met and peptidoglycan biosynthesis. Together, this study highlights the complexity of the interactions between a single amino acid, Met, and other bacterial processes leading to virulence in the host and indicates that disrupting the de novo biosynthetic pathway alone is likely to be ineffective as an antimicrobial therapy against S. Typhimurium.

A comparative analysis of sperm selection procedures prior to cryopreservation for Nili-Ravi buffalo bull (Bubalus bubalis) semen-: Assessment of its impact on post-thaw sperm functional quality.

Sperm selection techniques have been developed to get sperm suspensions enriched in motile and functional cells. Studies show that selection before cryopreservation improves post-thaw quality of cryopreserved sperm but information on buffalo bull sperm is scarce. The study was aimed to 1) perform a comparative analysis of sperm selection procedures; Swim-Up (SU), Sephadex™-G15 Filtration (S-G15) or Glass Wool Filtration (GWF) for total and motile cell recovery, 2) to assess the impact of sperm selection prior to cryopreservation on sperm quality (motility, morphology, cell membrane and normal apical ridge, viability and livability, chromatin integrity) and sperm functionality (Embryo Cleavage after IVF with selected sperm) in post-thawed suspensions of buffalo bull sperm. Semen was collected from 5 Nili Ravi buffalo bulls maintained at the Semen Production Unit Qadirabad, District Sahiwal, Pakistan. Ejaculates were divided into four aliquots for SU, S-G15 and GWF and control. After sperm selection, total and motile sperm recovery was highest in GWF samples (total sperm=84.08±8.39%; motile sperm=80.42±3.57%). An improvement (P<0.05) in all post-thaw parameters was observed in S-G15-selected sperm and, in some parameters in GWF-filtered sperm suspensions compared to control. The highest (P<0.05) embryo cleavage rate (%) was achieved with frozen-thawed sperm selected with S-G15 prior to cryopreservation (44.72±4.18) compared to control (21.98±3.00). In conclusion, post thaw sperm quality was improved after sperm selection from fresh buffalo bull semen through S-G15 and GWF procedures compared to SU and control while, the fertility rate (cleavage rate) was improved with sperm processed using the S-G15 procedure.