Validation of immunomodulatory effects of lipopolysaccharide through expression profiling of Th1 and Th2 biased genes in Newcastle disease virus vaccinated indigenous chicken.

BACKGROUND AND AIM: Newcastle disease (ND) is considered one of the most important poultry diseases with chicken morbidity and mortality rates up to 100%. Current vaccination programs allow the use of live attenuated vaccines in the field to protect against the disease, which alone is inefficient and requires repeat booster doses. Toll-like receptor agonists (e.g., lipopolysaccharide [LPS]) as adjuvants are the ones, most extensively studied and have shown to be very promising in delivering a robust balanced immune response. In the present study, we have evaluated the potential of LPS to elicit a strong immune response with respect to the elicitation of both Th1 (cell-mediated) and Th2 (humoral) immune arms. MATERIALS AND METHODS: A total of 72 apparently healthy 1-day-old indigenous unvaccinated chicks were randomly divided into six experimental Groups A to F (n=12). At 8-week of age chicks in Group A, C, and E were vaccinated with live attenuated La Sota strain ND vaccine along with LPS, bovine serum albumin, and normal saline solution, respectively, and those in Group B, D, and E were kept separately without vaccination. Sampling was done on days 0, 1, 3, 7, 14, 21, 35, and 60 after vaccination. After vaccination and respective adjuvant application, Th1 and Th2 cytokine expression were measured in mRNA of both blood and tissue samples. RESULTS: The results were validated by, hemagglutination inhibition and enzyme-linked immunosorbent assay tests, to check for the humoral as well as cell-mediated immune response in blood serum levels. The results showed an increase in mRNA expression of the Th1 biased cytokines in Group A (LPS+NDV) as compared to the control groups. Similar mRNA expression pattern was seen in blood as well as tissue samples. Validation of results also indicates an increase in Cell-mediated Immunity as well as a humoral immune response in Group A (LPS+NDV). CONCLUSION: The results of the study provided enough evidence to consider LPS as a potential vaccine adjuvants candidate against ND in chicken.

Applying the QUARTZ Trial Results in Clinical Practice: Development of a Prognostic Model Predicting Poor Outcomes for Non-small Cell Lung Cancers with Brain Metastases.

AIMS: The role of whole brain radiotherapy (WBRT) in patients with brain metastases from non-small cell lung cancers (NSCLC) has been questioned. However, no reliable criteria exist to identify patients who do not benefit from WBRT. The objective of the current study was to develop a prognostic model to identify such patients whose survival matches that of the Quality of Life after Treatment for Brain Metastases (QUARTZ) study. MATERIALS AND METHODS: Outcome data of patients with NSCLC with brain metastases undergoing WBRT enrolled in a prospective observational study in a tertiary cancer centre were used to develop a prognostic model. Baseline clinico-radiological factors were used for development of the model. The model was internally validated and calibration accuracy was checked for prediction of 70 day mortality. The generated prognostic model was presented as a nomogram. RESULTS: The median overall survival of 140 patients enrolled in the study was 166 days (95% confidence interval 108-242 days). The prognostic model identified gender, Karnofsky performance status and epidermal growth factor receptor activating mutation status as significant factors influencing overall survival. The model showed a modest discriminative ability with an optimism-corrected C-index of 0.64. However, model calibration error did reveal a moderate degree of calibration error. The high-risk subgroup identified by the model had a median overall survival of 67 days (95% confidence interval 56-101 days), which was similar to that observed in the QUARTZ trial. CONCLUSION: This prognostic model derived from traditional clinico-radiological features had a modest ability to identify patients with poor prognosis who may not benefit from WBRT. However, the high-risk subgroup identified using this prognostic model had a survival similar to that observed for patients in the QUARTZ trial.

14-3-3ζ binds the proteasome, limits proteolytic function and enhances sensitivity to proteasome inhibitors.

14-3-3 proteins are a family of master regulators of intracellular signaling, yet their impact on proteasome function is unknown. We demonstrate that 14-3-3ζ binds the 11S proteasome activator, limiting proteasome assembly and cellular capacity for protein degradation. To define the functional impact of 14-3-3ζ proteasomal binding in myeloma cells, silencing and overexpression experiments are performed. We find that downregulation of 14-3-3ζ impairs myeloma cell growth and confers resistance to clinically used proteasome inhibitors. In a large cohort of newly diagnosed myeloma patients, elevated expression of 14-3-3ζ is associated with high risk myeloma genetic subtypes and worse prognosis overall. Our work demonstrates the important role of 14-3-3ζ in regulating proteasome function, myeloma cell growth and sensitivity to therapeutics, and suggests regulation of 14-3-3ζ as a new approach in myeloma therapy.

Pronator teres accessorius: A new variant in flexor antebrachial musculature.

Muscles of flexor compartment of forearm have a common origin from medial epicondyle of humerus. Additional bellies of flexor muscles are commonly reported but presence of supernumerary muscles is an infrequent phenomenon. The present study describes an unusual muscle mass in flexor compartment of forearm simulating pronator teres. During routine dissection the upper limb of a 50 years old male cadaver, a supernumerary muscle was found on left side of the upper limb in the flexor compartment. The origin of the muscle was 2cm wide and aponeurotic in nature and attached to an oblique line extending from the inferior surface of the medial epicondyle and the medial surface of the trochlea. It was inserted on an oblique line 2.5cm wide on the radius in area between supinator superiorly and flexor digitorum profundus inferiorly. Existence of accessory muscles, which connect flexor muscles, could be explained embryologically by incomplete cleavage of flexor mass during development. The flexor muscles of the forearm develop from the flexor mass which subsequently divides into two layers: superficial and deep. The deep layer gives rise to flexor digitorum superficialis, flexor digitorum profundus and flexor pollicis longus. These supernumerary muscles are extremely rare entities and probably represent deranged embryological development or the process of atavism in which the anomalous part persist for a longer time in the tree of evolution.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences.

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo.

Cellular thermotolerance is independent of HSF 1 expression in zebu and crossbred non-lactating cattle.

Heat stress is an important domain of research in livestock due to its negative impact on production and disease resistance. The augmentation of stress in the body stimulates the antioxidative activity comprising various enzymes (viz., catalase, superoxide dismutase), metabolites (reduced glutathione, etc.), vitamins, minerals, etc. to combat the situation. The major key players involved in regulation of heat shock response in eukaryotes are the transcription factors, called as heat shock factors (HSF). They activate the heat shock protein (HSP) genes by binding to their promoters. Lymphocytes are considered to be the best model to evaluate the immunity in any living body as it contains plethora of white blood cells (WBCs).In this study, the peripheral blood mononuclear cells (PBMC) obtained from non-lactating Sahiwal vis-à-vis crossbred (Holstein Friesian × Sahiwal) cattle with 75% or more exotic inheritance were subjected to heat shock at 39, 41, and 43 °C in three different incubators, in vitro. The cell count and viability test of pre and post heat stress of concerned PBMCs indicated that the crossbreeds are more prone to heat stress as compared to Sahiwal. The reverse transcription PCR (qRT-PCR) expression data revealed an increment in HSF1 expression at 41 °C which subsequently declined (non-significantly) at 43 °C in both breeds post 1 h heat shock. However, the association between the HSF 1 expression and antioxidative activity through correlation analysis was found to be non-significant (P < 0.05), though enzymatic activity appeared to behave in a similar fashion in both breeds at 5% level of significance (P < 0.05). This rule out the role of HSF1 expression level on the activity of enzymes involved in oxidative stress in vitro in zebu and crossbred cattle.

Biocomputational analysis of evolutionary relationship between toll-like receptor and nucleotide-binding oligomerization domain-like receptors genes.

AIM: The active domains (TIR and NACHT) of the pattern recognition receptors (PRRs: Toll-like receptors [TLRs] and nucleotide-binding oligomerization domain [NOD]-like receptors [NLR], respectively) are the major hotspots of evolution as natural selection has crafted their final structure by substitution of residues over time. This paper addresses the evolutionary perspectives of the TLR and NLR genes with respect to the active domains in terms of their chronological fruition, functional diversification, and species-specific stipulation. MATERIALS AND METHODS: A total of 48 full-length cds (and corresponding peptide) of the domains were selected as representatives of each type of PRRs, belonging to divergent animal species, for the biocomputational analyses. The secondary and tertiary structure of the taurine TIR and NACHT domains was predicted to compare the relatedness among the domains under study. RESULTS: Multiple sequence alignment and phylogenetic tree results indicated that these host-specific PRRs formed entirely different clusters, with active domains of NLRs (NACHT) evolved earlier as compared to the active domains of TLRs (TIR). Each type of TLR or NLR shows comparatively less variation among the animal species due to the specificity of action against the type of microbes. CONCLUSION: It can be concluded from the study that there has been no positive selection acting on the domains associated with disease resistance which is a fitness trait indicating the extent of purifying pressure on the domains. Gene duplication could be a possible reason of genesis of similar kinds of TLRs (virus or bacteria specific).

Hierarchical patterning of hydrogels by replica molding of impregnated breath figures leads to superoleophobicity.

The surface chemistry and topography govern the spreading of liquids on a solid. When an oil drop makes a contact angle, θ > 90° on a solid surface, the solid is termed as oleophobic. Adding roughness to an inherently oleophobic surface enhances its oil dewetting and can lead to superoleophobicity when θ > 150°. In this study, we introduce the concept of a two-tier hierarchical roughness on the surface of soft materials such as hydrogels by forming the patterned inverse replica of breath figure polymer films impregnated with nanoparticles. The directed deposition of nanoparticles in the breath figure pores is accomplished by an aerosol assisted technique that exclusively leads to deposition within the pores and filling of the pores. The inverse replica of such impregnated films exhibits a close packed hexagonally structured second tier of surface roughness which directly leads to a superoleophobic surface. Since these structures have well defined geometries, it is possible to estimate the contact angle by assuming a partial wetting of the oil drop in a 'fakir' state on the rough surface. The estimation is in good agreement with the experimental contact angle value. While the work demonstrates a facile method to impart superoleophobicity to a hydrogel surface, it also demonstrates new methods to imbue breath figure pores with functional materials that can be easily transferred to the pores of the inverse replica.

Handmade cloning: recent advances, potential and pitfalls.

Handmade cloning (HMC) is the most awaited, simple and micromanipulator-free version of somatic cell nuclear transfer (SCNT). The requirement of expensive micromanipulators and skilled expertise is eliminated in this technique, proving it as a major revolution in the field of embryology. During the past years, many modifications have been incorporated in this technique to boost its efficiency. This alternative approach to micromanipulator based traditional cloning (TC) works wonder in generating comparable or even higher birth rates in addition to declining costs drastically and enabling cryopreservation. This technique is not only applicable to intraspecies nuclear transfer but also to interspecies nuclear transfer (iSCNT) thus permitting conservation of endangered species. It also offers unique possibilities for automation of SCNT which aims at production of transgenic animals that can cure certain human diseases by producing therapeutics hence, providing a healthier future for the wellbeing of humans. The present review aims at highlighting certain aspects of HMC including recent advancements in procedure and factors involved in elevating its efficiency besides covering the potentials and pitfalls of this technique.

Adductor accessorius - an unusual supernumerary adductor muscle of thigh.

The knowledge of variant anatomy of medial compartment of thigh is extremely important in surgical procedures requiring obturator nerve block. The nerve block is also recommended for providing additional analgesia following major knee surgeries and for relieving painful adductor muscle spasm. The interfascial injection technique is commonly followed in ultrasound guided obturator nerve block. For this procedure it is imperative to identify the adductor muscles on sonography to inject the anesthetic solution in the intermuscular fascial planes. The presence of an additional muscle can prove useful in myocutaneous flap surgeries for reconstruction especially of perineum. We hereby report a case of such an anomalous supernumerary adductor muscle present between adductor brevis and proximal part of adductor magnus, in an adult Indian male cadaver, recorded during routine dissection in Department of Anatomy, Vardhman Mahavir Medical College and Safdarjung Hospital. The muscle was innervated by the posterior division of obturator nerve. The morphology, embryological basis and clinical aspects are also discussed.

Rare atypical vascular pattern of subscapular artery: Anatomico-clinical insight.

BACKGROUND AND OBJECTIVE: The use of muscles of scapular region in transposition and reparative surgery has made the anatomical knowledge of axillary artery and its branches imperative. The subscapular arterial tree is frequently used as a source of microvascular grafts to replace damaged or diseased portion of arteries especially in upper and lower limbs. This investigation is aimed to study the variation in branching pattern of the third part of axillary artery. MATERIALS AND METHODS: Routine educational cadaveric dissection of a young adult Indian male cadaver. RESULTS: The present article highlights a rare variation of the subscapular artery and its branches. The subscapular artery displayed a unique pattern of trifurcation. Its branches were seen supplying serratus anterior, subscapularis, latissimus dorsi and teres major muscles. CONCLUSIONS: The study focuses on a new variation of the subscapular artery which could prove useful for interventional radiologists and surgeons in reconstructive surgeries.

Spatially directed vesicle capture in the ordered pores of breath-figure polymer films.

This work describes a new method to selectively capture liposomes and other vesicle entities in the patterned pores of breath-figure polymer films. The process involves the deposition of a hydrophobe containing biopolymer in the pores of the breath figure, and the tethering of vesicles to the biopolymer through hydrophobic interactions. The process is versatile, can be scaled up and extended to the deposition of other functional materials in the pores of breath figures.

Unusual branching pattern of brachial artery - Embryological basis and clinicoanatomical insight.

Variations in the arterial pattern of upper limb are of colossal importance to the surgeons as they are liable to iatrogenic injuries. During routine dissection for undergraduate medical students, an anomaly of brachial artery was discovered. The brachial artery terminated at higher level into ulnar and radial artery. The common interosseus artery took origin arising from radial artery. The ulnar artery did not give any branches in the forearm. Both radial and ulnar artery displayed a superficial course in the forearm. The anatomical knowledge of these variations may be of great help for the clinicians in planning and conducting flap harvesting during reconstructive surgeries and in arteriography.

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay.

PURPOSE: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. MATERIALS AND METHODS: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. RESULTS: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. CONCLUSION: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

Combined spinal-epidural for labor analgesia with low-dose bupi- vacaine but without any opioid in the spinal component: can we improve upon the traditional?

AIM: To compare the efficacy and safety of combined spinal-epidural (CSE, with the spinal component using low-dose heavy bupivacaine without opioid) with low-dose epidural analgesia in labor. METHODS: Sixty consenting adult parturients requesting epidural were randomly allocated to two equal groups to receive either low-dose epidural or CSE analgesia. The Epidural Group received 10ml of a mixture of 0.1% bupivacaine and 2 µg/ml fentanyl, followed by a 6 ml first bolus after 10 min if needed (not considered as a top-up). All patients received 8 ml/h continuous background infusion, with patient-controlled top-up bolus of 6 ml with a lock-out interval of 12 min. In the CSE Group, 0.5% (heavy) bupivacaine 2.5 mg was used to produce spinal block (without concomitant use of any opioid), followed by epidural continuous background infusion of 8 ml of 0.1% of bupivacaine and 2 µg/ml of fentanyl, with a similar top-up provision as in the Epidural Group. Primary outcome was total drug consumption during labor, corrected by the duration of labor (ml/h). RESULTS: Mean total drug consumption during labor was significantly less in the CSE group (9.69 ml/h) than in Epidural group (13.52 ml/h ; P < 0.00 1). CSE also led to significantly faster onset of analgesia and sensory block. There was no significant difference between the two groups with regard to all other variables. Maternal satisfaction was rated as 'excellent' by all subjects. CONCLUSIONS: CSE using low-dose heavy bupivacaine without opioid required less drug consumption produced faster onset of pain control and sensory block than low-dose epidural analgesia. There were no other significant inter-group differences.

Liposomes tethered to a biopolymer film through the hydrophobic effect create a highly effective lubricating surface.

Liposomal coatings are formed on films of a biopolymer, hydrophobically modified chitosan (hm-chitosan), containing dodecyl groups as hydrophobes along the polymer backbone. The alkyl groups insert themselves into the liposome bilayer through hydrophobic interactions and thus tether liposomes, leading to a densely packed liposome layer on the film surface. Such liposomal surfaces exhibit effective lubrication properties due to their high degree of hydration, and reduce the coefficient of friction to the biologically-relevant range. The compliancy and robustness of these tethered liposomes allow retention on the film surface upon repeated applications of shear. Such liposome coated films have potential applications in biolubrication.

Xpert(®) MTB/RIF assay for tuberculosis diagnosis: evaluation in an Indian setting.

The present study was conducted to evaluate the performance of the Xpert(®) MTB/RIF assay and compare Xpert results with solid and MGIT 960 liquid culture system. A total of 134 patients who had failed the Category I or II regimen were recruited for evaluation. Xpert correctly identified all Mycobacterium tuberculosis isolates. The sensitivity and specificity of the Xpert assay for the detection of rifampicin resistance was respectively 98.2% and 97.0% when compared with MGIT 960 results.

Phylogenetic associations with drug-resistant Mycobacterium tuberculosis isolates in a paediatric population.

SETTING: Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. OBJECTIVES: As paediatric tuberculosis (TB) is a surrogate marker for actively transmitted disease in a community, we investigated drug resistance patterns of 97 Mycobacterium tuberculosis complex strains isolated from children and explored their phylogenetic associations. DESIGN: A total of 111 paediatric patients who attended the out-patient department during the study period 2009-2011 and whose sputum samples were sent to the Microbiology Department for liquid culture and drug susceptibility testing (DST) were included in this study. DST and spoligotyping were performed on cultures positive for M. tuberculosis complex. RESULTS: DST against four first-line drugs showed that 31 of 97 (32%) strains were pan-susceptible, while 66/97 (68%) were resistant to at least one drug, including 55/97 (56.7%) that were resistant to at least isoniazid and rifampicin (i.e., multidrug-resistant). The majority of the isolates (n = 81/90, 90%) belonged to the principal genetic group 1 strains, the most predominant spoligotyping clusters being spoligotyping international type (SIT)1/Beijing (n = 28), SIT26/CAS1-Delhi (n = 27) and SIT53/T1 (n = 6). CONCLUSION: The involvement of Beijing and CAS1-Delhi clades in paediatric TB patients suggests that these two lineages play a major role in ongoing active transmission.

A rare conglomeration of neurovascular variations in the brachium-antebrachium: clinico-embryological elucidation.

During routine dissection in left upper limb in an elderly male cadaver, variations in the arterial and neural pattern were noticed. The undersized brachial artery divided terminally into radial and ulnar artery at a distance of 15.4 cm from the tip of coracoid process, proximal to its usual division in cubital fossa. Additionally, the superior and inferior ulnar collateral artery instead of arising from the brachial artery, emanated from ulnar artery. The course of the median nerve also deserves a brief mention as it crossed the brachial artery from medial to lateral side to position itself laterally in the midbrachium. Coursing further, it shifted from lateral to medial side over the ulnar artery in the distal third of arm, thus placing itself medial to ulnar artery in the cubital fossa. There was also a communication between a medial offshoot of the musculocutaneous nerve and the median nerve 3 cm from the bifurcation of the brachial artery. Although the variations in the origin, termination, course and branching pattern of upper limb vessels and nerves are well documented, but the multitude of disparity in morphology with confusing neurovascular assembly put together in the same limb denotes an outstanding case. An attempt has been made to correlate the present anatomical variations with embryological aspects. Moreover, the clinical inferences of such variations are discussed for planning, performing and analyzing invasive and noninvasive clinical procedures in this region.