Bioselective and Radiopaque Zinc-Biopolymeric Complex-Based Porous Biomaterials Promote Mammalian Tissue Ingrowth In Vivo While Inhibiting Microbial Biofilm Gene Expression and Biofilm Formation.

Metal-organic complexes have shown astounding bioactive properties; however, they are rarely explored as biomaterials. Recent studies showed that carboxymethyl-chitosan (CMC) genipin-conjugated zinc biomimetic scaffolds have unique bioselective properties. The biomaterial was reported to be mammalian cell-friendly; at the same time, it was found to discourage microbial biofilm formation on its surface, which seemed to be a promising solution to addressing the problem of trauma-associated biofilm formation and development of antimicrobial resistance. However, the mechanically frail characteristics and zinc overload raise concerns and limit the potential of the said biomaterials. Hence, the present work is focused on improving the strength of the earlier scaffold formulations, testing its in vivo efficacy and reaffirming its action against biofilm-forming microbe Staphylococcus aureus. Scaling up of CMC proportion increased rigidity, and 8% CMC was found to be the ideal concentration for robust scaffold fabrication. Freeze-dried CMC scaffolds with or without genipin (GP) cross-linking were conjugated with zinc using 2 M zinc acetate solution. Characterization results indicated that the CMC-Zn scaffolds, without genipin, showed mechanical properties close to bone fillers, resist in vitro enzymatic degradation until 4 weeks, are porous in nature, and have radiopacity close to mandibular bones. Upon implantation in a subcutaneous pocket of Wistar rats, the scaffolds showed tissue in-growth with simultaneous degradation without any signs of toxicity past 28 days. Neither were there any signs of toxicity in any of the vital organs. Considering many superior properties among the other formulations, the CMC-Zn scaffolds were furthered for biofilm studies. CMC-Zn showed negligible S. aureus biofilm formation on its surface as revealed by an alamar blue-based study. RT-PCR analysis revealed that CMC-Zn downregulated the expression of pro-biofilm effector genes such as icaC and clfB. A protein docking study predicted the inhibitory mechanism of CMC-Zn. Although it binds strongly when alone, at high density, it may cause inactivation of the transmembrane upstream activators of the said genes, thereby preventing their dimerization and subsequent inactivation of the effector genes. In conclusion, zinc-conjugated carboxymethyl-chitosan scaffolds are mechanically robust, porous, yet biodegradable, harmless to the host in the long term, they are radiopaque and prevent biofilm gene expression in notorious microbes; hence, they could be a suitable candidate for bone filler applications.

Development of guar gum reinforced calcium magnesium phosphate-based bone biocement.

This study aims at developing a calcium magnesium phosphate-based bone biocement that combines a natural polymer and regenerative properties of bone bonding materials. The formulation of this biocement consists of oxidized guar gum, polydopamine, and calcium magnesium phosphate. The oxidized guar gum is easily soluble in water and has a slightly basic pH, unlike unmodified guar gum, thus allowing a homogenous paste to form in the alkaline environment of calcium magnesium phosphate. Three different oxidized degrees of guar gum were made, and the impact on the biocement properties was studied. The modified guar gum-reinforced biocement (OGG C2) displayed higher mechanical strength and lower degradation rates than OGG B1 and OGG A0. Furthermore, samples with polydopamine exhibited better results, thus, improving the already reinforced biocement. Morphological studies of the biocement displayed a highly porous structure with porosity varying among biocement containing different oxidized guar gum and polydopamine levels.

Comparative study of hydroxyapatite synthesized using Schiff base and wet chemical precipitation methods.

Hydroxyapatite (HAp) exists as an inorganic and crystalline composition present in bones and dental enamel, and hence can be utilized as a direct element or as part of the composition of biomaterials and implants for dental and orthopaedic applications. Listed below are a few synthesis techniques for HAp that are listed in the literature: solid-state and mechano-chemical methods (dry methods), wet chemical precipitation and sol-gel methods (wet methods), and combustion and pyrolysis methods (high-temperature processes). Nevertheless, there are new and more productive techniques that result in HAp with a regulated morphology, such as the Schiff base method, which, on reaction with calcium and phosphate precursors, forms chelating complexes to produce HAp nuclei. This research paper presents the comparison in characteristics between HAp synthesized using Schiff base (HAp-SB), wet chemical precipitation (HAp-WC) methods, and commercial HAp (HAp-CM) in their powdered and pelleted form. The average size of HAp-WC particles in the spherical form was found to be 603 nm ± 176, HAp-SB were found to have rod-like morphology, which is very similar to human bone-like HAp, with an average length and width of 1522 nm ± 759 and 400 nm ± 112, respectively, and HAp-CM were found to have spherical morphology with dimensions of 52 nm ± 25. Biological studies show that cell viability of HAp-SB pellet (202.01% ± 8.16) seemed to have higher cell proliferation properties than HAp-WC pellet (145.7% ± 5.11) and HAp-CM pellet (71.53% ± 3.61) due to its higher aspect ratio, and hence higher surface area for the cells to adhere. In a detailed study, it is observed that both techniques had their advantages, and there were no significant disadvantages observed.

RNA binding protein IGF2BP1 synergizes with ETV6-RUNX1 to drive oncogenic signaling in B-cell Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein. METHODS: Gene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model. RESULTS: We observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance. IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways. Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis. CONCLUSION: Our data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.

Investigation of Giant Cell Tumor of Bone and Tissue Engineering Approaches for the Treatment of Giant Cell Tumor of Bone.

Primary bone tumors such as Ewing sarcoma, osteosarcoma, and chondrosarcoma, secondary bone tumors developed from progressive malignancies, and metastasized bone tumors are more prevalent and studied descriptively through biology and medical research. Less than 0.2% of cancer diagnoses are caused by rare bone-originating tumors, which despite being rare are particularly difficult due to their high death rates and substantial disease burden. A giant cell tumor of bone (GCTB) is an intramurally invasive but rare and benign type of bone tumor, which seldom metastasizes. The most often prescribed medication for GCTB is Denosumab, a RANKL (receptor activator of nuclear factor κB ligand) inhibitor. Because pharmaceutical drug companies rely on two-dimensional and animal models, current approaches for investigating the diverse nature of tumors are insufficient. Cell line based medication effectiveness and toxicity studies cannot predict tumor response to antitumor medicines. It has already been investigated in detail why molecular pathways do not reproduce in vitro, a phenomenon known as flat biology. Due to physiological differences between human beings and animals, animal models do not succeed in identifying side effects of the treatment, emulating metastatic growth, and establishing the link between cancer and the immune system. This review summarizes and discusses GCTB, the disease, its cellular composition, various bone tumor models, and their properties and utilization in research. As a result, this study delves deep into in vitro testing, which is vital for scientists and physicians in various fields, including pharmacology, preclinical investigations, tissue engineering, and regenerative medicine.

IGF2BP3 as a Prognostic Biomarker in Well-Differentiated/Dedifferentiated Liposarcoma.

BACKGROUND: Although IGF2BP3 has been implicated in tumorigenesis and poor outcomes in multiple cancers, its role in soft-tissue sarcoma (STS) remains unknown. Preliminary data have suggested an association with IGF2BP3 expression among patients with well-differentiated/dedifferentiated liposarcoma (WD/DD LPS), a disease where molecular risk stratification is lacking. METHODS: We examined the survival associations of IGF2BP3 via univariate and multivariate Cox regression in three unique datasets: (1) the Cancer Genome Atlas (TCGA), (2) an in-house gene microarray, and (3) an in-house tissue microarray (TMA). A fourth dataset, representing an independent in-house TMA, was used for validation. RESULTS: Within the TCGA dataset, IGF2BP3 expression was a poor prognostic factor uniquely in DD LPS (OS 1.6 vs. 5.0 years, p = 0.009). Within the microarray dataset, IGF2BP3 expression in WD/DD LPS was associated with worse survival (OS 7.7 vs. 21.5 years, p = 0.02). IGF2BP3 protein expression also portended worse survival in WD/DD LPS (OS 3.7 vs. 13.8 years, p < 0.001), which was confirmed in our validation cohort (OS 2.7 vs. 14.9 years, p < 0.001). In the multivariate model, IGF2BP3 was an independent risk factor for OS, (HR 2.55, p = 0.034). CONCLUSION: IGF2BP3 is highly expressed in a subset of WD/DD LPS. Across independent datasets, IGF2BP3 is also a biomarker of disease progression and worse survival.

Scaffold-based spheroid models of glioblastoma multiforme and its use in drug screening.

Among several types of brain cancers, glioblastoma multiforme (GBM) is a terminal and aggressive disease with a median survival of 15 months despite the most intensive surgery and chemotherapy. Preclinical models that accurately reproduce the tumor microenvironment are vital for developing new therapeutic alternatives. Understanding the complicated interactions between cells and their surroundings is essential to comprehend the tumor's microenvironment, however the monolayer cell culture approach falls short. Numerous approaches are used to develop GBM cells into tumor spheroids, while scaffold-based spheroids provides the opportunity to investigate the synergies between cells as well as cells and the matrix. This review summarizes the development of various scaffold-based GBM spheroid models and the prospective for their use as drug testing systems.

Dual drug delivery platforms for bone tissue engineering.

The dual delivery platforms used in bone tissue engineering provide supplementary bioactive compounds that include distinct medicines and growth factors thereby aiding enhanced bone regeneration. The delivery of these compounds can be adjusted for a short or prolonged time based on the requirement by altering various parameters of the carrier platform. The platforms thus used are fabricated to mimic the niche of the bone microenvironment, either in the form of porous 3D structures, microspheres, or films. Thus, this review article focuses on the concept of dual drug delivery platform and its importance, classification of various platforms for dual drug delivery specific to bone tissue engineering, and finally highlights the foresight into the future direction of these techniques for better clinical applications.

The RNA-binding protein IGF2BP3 is critical for MLL-AF4-mediated leukemogenesis.

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.

Unveiling the secrets of marine-derived fucoidan for bone tissue engineering-A review.

Biomedical uses for natural polysaccharides of marine origin are growing in popularity. The most prevalent polysaccharides, including alginates, agar, agarose and carrageenan, are found in seaweeds. One among these is fucoidan, which is a sulfated polysaccharide derived from brown algae. Compared to many of the biomaterials of marine origin currently in research, it is more broadly accessible and less expensive. This polysaccharide comes from the same family of brown algae from which alginate is extracted, but has garnered less research compared to it. Although it was the subject of research beginning in the 1910's, not much has been done on it since then. Few researchers have focused on its potential for biomedical applications; nevertheless, a thorough knowledge of the molecular mechanisms behind its diverse features is still lacking. This review provides a quick outline of its history, sources, and organization. The characteristics of this potential biomaterial have also been explored, with a thorough analysis concentrating on its use in bone tissue engineering. With the preclinical research completed up to this point, the fucoidan research status globally has also been examined. Therefore, the study might be utilized as a comprehensive manual to understand in depth the research status of fucoidan, particularly for applications related to bone tissue engineering.

A Review on Development of Bio-Inspired Implants Using 3D Printing.

Biomimetics is an emerging field of science that adapts the working principles from nature to fine-tune the engineering design aspects to mimic biological structure and functions. The application mainly focuses on the development of medical implants for hard and soft tissue replacements. Additive manufacturing or 3D printing is an established processing norm with a superior resolution and control over process parameters than conventional methods and has allowed the incessant amalgamation of biomimetics into material manufacturing, thereby improving the adaptation of biomaterials and implants into the human body. The conventional manufacturing practices had design restrictions that prevented mimicking the natural architecture of human tissues into material manufacturing. However, with additive manufacturing, the material construction happens layer-by-layer over multiple axes simultaneously, thus enabling finer control over material placement, thereby overcoming the design challenge that prevented developing complex human architectures. This review substantiates the dexterity of additive manufacturing in utilizing biomimetics to 3D print ceramic, polymer, and metal implants with excellent resemblance to natural tissue. It also cites some clinical references of experimental and commercial approaches employing biomimetic 3D printing of implants.

Self-Healing Hydrogels: Preparation, Mechanism and Advancement in Biomedical Applications.

Polymeric hydrogels are widely explored materials for biomedical applications. However, they have inherent limitations like poor resistance to stimuli and low mechanical strength. This drawback of hydrogels gave rise to ''smart self-healing hydrogels'' which autonomously repair themselves when ruptured or traumatized. It is superior in terms of durability and stability due to its capacity to reform its shape, injectability, and stretchability thereby regaining back the original mechanical property. This review focuses on various self-healing mechanisms (covalent and non-covalent interactions) of these hydrogels, methods used to evaluate their self-healing properties, and their applications in wound healing, drug delivery, cell encapsulation, and tissue engineering systems. Furthermore, composite materials are used to enhance the hydrogel's mechanical properties. Hence, findings of research with various composite materials are briefly discussed in order to emphasize the healing capacity of such hydrogels. Additionally, various methods to evaluate the self-healing properties of hydrogels and their recent advancements towards 3D bioprinting are also reviewed. The review is concluded by proposing several pertinent challenges encountered at present as well as some prominent future perspectives.

Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia.

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

Effectiveness in Bowel Cleansing and Patient Tolerability of Polyethylene Glycol versus Sodium Picosulphate in Patients Undergoing Colonoscopy.

INTRODUCTION: Colonoscopy is considered as a gold standard investigation for screening of colorectal cancer and other lower gastrointestinal pathologies. Adequate bowel preparation is absolutely necessary for a fruitful colonoscopy. Various bowel cleansing agents are being used for his purpose. The aim of the present study was to compare the two bowel cleansing agents: a single dose of Polyethylene Glycol (PEG) solution and a split dose of Sodium Picosulfate (Na PICOSUL) tablet with regards to cleansing efficacy and tolerability among the patients scheduled for colonoscopy. METHODS: It is an open-label hospital-based observational study. A total of sixty-four patients were grouped randomly into two groups of bowel cleansing agents that are PEG and Na PICOSUL during the study period between 1st December 2015 and 30th November 2016. Patients' tolerability was evaluated using a structured questionnaire, and the bowel cleansing efficacy was evaluated using the Aronchick Bowel Preparation Scale (ABPS). RESULTS: The group that received PEG solution was found to have better efficacy than that which received Na PICOSUL tablet (63.3% versus 29.4%, respectively, with a P value < 0.028) with excellent grade as per ABPS. The Na PICOSUL group was found better in terms of tolerability than the PEG group as nausea/vomiting was encountered significantly higher in the PEG group than in the Na PICOSUL group (43.3% versus 11.8%, respectively, with a P value < 0.01). CONCLUSIONS: Colonic preparation with a split dose of Na PICOSUL tablet was better tolerated than the evening before regimen of PEG solution. However, PEG solution was found to be more efficacious in bowel cleansing, but procedural performance and lesion detection were similar for both agents.

Crosslinking of gum-based composite scaffolds for enhanced strength and stability: A comparative study between sodium trimetaphosphate and glutaraldehyde.

Tissue engineering is one of the potential fields in the domain of regenerative medicine. Engineered scaffolds are an excellent substitute for the conventional use of bone grafts as they are biocompatible, economic, and provide limitless supply with no risk of disease transmission. Gum-based scaffolds present a good scope for studying tissue-engineering models and analyzing controlled drug delivery. Uniform blending of the gums and the presence of the optimal concentration of appropriate crosslinkers are very crucial for biodegradability nature. Gum-based scaffolds containing gellan gum, xanthan gum, polyvinyl alcohol, and hydroxyapatite, cross-linked with either glutaraldehyde (GA) or sodium trimetaphosphate (STMP) were fabricated to study the efficiency of crosslinkers and were characterized for degradation profile, swelling capacity, porosity, mechanical strength, morphology, X-ray diffraction, Fourier-transform infrared, and in vitro biocompatibility. Scaffolds crosslinked with STMP exhibited higher degradation rate at Day 21 than scaffolds crosslinked with GA. However, higher compressive strength was obtained for scaffolds cross-linked with STMP signifying that they have a better ability to resist compressive forces. Superior cell viability was observed in STMP-crosslinked scaffolds. In conclusion, STMP serves as a better crosslinker in comparison to GA and can be used in the fabrication of scaffolds for bone tissue engineering.

Effect of interpolymer complex formation between chondroitin sulfate and chitosan-gelatin hydrogel on physico-chemical and rheological properties.

The present study focuses on the fabrication of three-dimensional (3D) natural polymeric hydrogel containing chitosan (C), gelatin (GE) and chondroitin sulfate A (CS) to surpass the drawbacks of chitosan-gelatin (C-GE) hydrogel. Hydrogels were prepared using dual gelling agents in the form of ß-glycerol phosphate (ß-GP) and sodium hydrogen carbonate (SHC) and failed to gel in the absence of either gelling agent. All the hydrogels showed the ability to self-heal when broken into two parts. The addition of CS resulted in the formation of elastic hydrogels during the entire range of applied shear strain (0.001-100 %) compared to hydrogel without CS. CS containing hydrogels resulted in 25-41 % bovine serum albumin in vitro release compared to 77 % in hydrogel without CS at the end of 16 days. The addition of VEGF165 to hydrogel improved cell proliferation drastically in C-GE hydrogel. C-GE-CS hydrogels exhibited better cell growth compared to C-GE hydrogel with or without the addition of VEGF165.

Quantitative Evaluation in Estimating Sources Underlying Brain Oscillations Using Current Source Density Methods and Beamformer Approaches.

Brain oscillations from EEG and MEG shed light on neurophysiological mechanisms of human behavior. However, to extract information on cortical processing, researchers have to rely on source localization methods that can be very broadly classified into current density estimates such as exact low-resolution brain electromagnetic tomography (eLORETA), minimum norm estimates (MNE), and beamformers such as dynamic imaging of coherent sources (DICS) and linearly constrained minimum variance (LCMV). These algorithms produce a distributed map of brain activity underlying sustained and transient responses during neuroimaging studies of behavior. On the other hand, there are very few comparative analyses that evaluates the "ground truth detection" capabilities of these methods. The current article evaluates the reliability in estimation of sources of spectral event generators in the cortex using a two-pronged approach. First, simulated EEG data with point dipoles and distributed dipoles are used to validate the accuracy and sensitivity of each one of these methods of source localization. The abilities of the techniques were tested by comparing the localization error, focal width, false positive (FP) ratios while detecting already known location of neural activity generators under varying signal-to-noise ratios (SNRs). Second, empirical EEG data during auditory steady state responses (ASSRs) in human participants were used to compare the distributed nature of source localization. All methods were successful in recovery of point sources in favorable signal to noise scenarios and could achieve high hit rates if FPs are ignored. Interestingly, focal activation map is generated by LCMV and DICS when compared to eLORETA while control of FPs is much superior in eLORETA. Subsequently drawbacks and strengths of each method are highlighted with a detailed discussion on how to choose a technique based on empirical requirements.

Development of mechanically compliant 3D composite scaffolds for bone tissue engineering applications.

Cancellous bone region of the human native bone has the potential to bear significant mechanical loads. However, due to the following parameters such as low trabecular volume, more porosity, less thickness and more interconnectivity, cancellous bone is accessible to damage in accidents or when aging. This research led to the effective fabrication and engineering of cancellous region of the bone for its application in reconstruction or filling of bone voids after resection of large tumor mass. Scaffolds containing hydroxyapatite (HAp), polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) (5% polymer and HAp concentrations) at different HAp: polymer composite ratios (50:50, 70:30 and 80:20), were fabricated by freeze-drying method using only water as solvent and ribose as crosslinker for the scaffolds. The fabricated scaffolds were characterized for its mechanical (compressive) strength, chemical properties using FT-IR and XRD analyses, swelling and degradation studies, confirmation of mineralization process by immersion in simulated body fluid (SBF) for 7, 15 and 30 days, morphological analysis using scanning electron microscopy (SEM), and biocompatibility properties by performing MTT analysis. From the combined interpretation of the above mentioned tests, it was proven that 80:20 ratio of HAp to polymers was found to be the most suitable scaffold in terms of its optimal properties for its use as bone graft material for trabecular bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3267-3274, 2018.

First report of molecular characterization and sequence phylogenetic analysis of Linguatula serrata from India.

Linguatula serrata is a pentastomid which is worldwide in distribution. However, a very few references are reported from India. In the present study, the cox I gene of L. serrata nymphs, originally isolated from mesenteric lymph nodes of buffaloes, was amplified and custom sequenced. Based on sequence analysis, two haplotypes were noticed and were subsequently submitted in NCBI database. The sequences were also compared with the other sequences available in the pubmed and phylogenetic analysis coupled with nucleotide homologies were commutated. The studied Indian isolates were found closer to Bangladesh and Iran isolates. This is the first report of molecular characterization of L. serrata from India.

Diagnostic potential of low molecular weight excretory secretory proteins of Paramphistomum epiclitum for caprine amphistomosis.

In the present study, the 75% alcoholic fractionation of excretory-secretory (ES) antigen isolated from 200 to 300 live P. epiclitum was assessed for its diagnostic potential for the detection of caprine amphistomosis by using antibody detection enzyme immunoassay. Prior to enzyme immunoassay, 75% alcoholic fractionation of excretory-secretory (ES) antigen was subjected to SDS- PAGE and western blot analysis for the presence of immunoreactive polypeptides. SDS-PAGE analysis of ES antigen resolved a total 7 polypeptides bands of size 56, 27, 25, 22.5, 12, 11 and 10 kDa. Western blot analysis revealed only two immunoreactive polypeptides (11 kDa and 12 kDa) when polypeptides resolved in SDS-PAGE were probed with known positive pooled serum. None of the polypeptides showed reactions with pooled known negative serum. The working dilutions of antigen, sera and conjugates were determined by checkerboard titration for employing ELISA and cut-off O.D. was calculated 0.616 by using the mean absorbance value of 11 negative kid sera. The sensitivity and specificity of ELISA was found to be 100% and 86.76%, respectively. As per kappa value estimation, the strength of agreement was found to be good. Antibodies to 75% alcoholic fractionation of ES antigen was detected in 20% goats (n = 160) of either sex, although faecal examination detected 10.6% goats to be infected with amphistomosis. The study confirmed that 75% alcoholic fractionation of ES antigen of P. epiclitum based ELISA had good value for serodiagnosis of caprine amphistomosis.