How do root fungi of Alnus nepalensis and Schima wallichii recover during succession of abandoned land?

Alnus nepalensis and Schima wallichii are native tree species accompanying succession in abandoned agricultural land in the middle mountainous region of central Nepal. To understand how root fungi recover during spontaneous succession, we analyzed the diversity and composition of arbuscular mycorrhizal (AM), ectomycorrhizal (ECM), and total fungi in tree fine roots from three land use types, short-term abandoned land (SA), long-term abandoned land (LA), and regenerated forest (RF) as a reference. Additionally, ECM morphotypes were examined. The results showed different speeds of succession in the studied fungal groups. While the change in the AM fungal community appears to be rapid and LA resembles the composition of RF, the total fungi in the abandoned land types are similar to each other but differed significantly from RF. Interestingly, the relative abundance of Archaeosporaceae followed a trend differing between the tree species (SA < LA in A. nepalensis, but SA > LA in S. wallichii). Unlike AM and total fungi, there was no significant difference in the ECM community of A. nepalensis between land use types, probably due to their low species diversity (9 ECM morphotypes, 31 ECM operational taxonomic units). However, Cortinarius sp. was significantly more abundant in RF than in the other land use types, whereas Alnicola, Tomentella, and Russula preferred young stages. Our results suggest that for both studied tree species the AM fungal succession could reach the stage of regenerated forest relatively fast. In the case of total fungi, because of hyperdiversity and composed of species specialized to a variety of environments and substrates, the transition was expected to be delayed in abandoned land where the vegetation was still developing and the ecosystem was not as complex as that found in mature forests.

Endotracheal Intubation at 3,600 Meters Above Sea Level for High-Altitude Pulmonary Edema Followed by Helicopter Evacuation in Nepal.

Ranging from 64 to 8848 m above sea level, Nepal is a country rich in hilly and mountainous terrain.1 24.8% of Nepal's land area is above 3000 m, 18.9% is between 3000 and 5000 m, and 5.9% is above 5000 m.2 Hikers and trekkers are increasingly attracted to this challenging altitude and terrain, which presents risks for altitude sickness and other physical complications. Responding to medical emergencies in high-altitude areas in Nepal is highly challenging. This difficulty is often exacerbated by inclement weather, unavailability of helicopters, and poor communication regarding the location and condition of patients requiring medical attention and evacuation. High-altitude pulmonary edema (HAPE) is an illness characterized by non-cardiogenic pulmonary edema, which occurs not infrequently in individuals who rapidly ascend above 2500-3000 m in elevation,3 and which has a high mortality rate if not treated in a timely manner. Improved outcomes would be likely if skilled and equipped medical staff had better access to the sites of high-altitude expeditions in Nepal, so that life-saving interventions could be performed promptly. We report the case of a patient with HAPE who was intubated in the field at an altitude of 3600 m, and then evacuated via helicopter to a healthcare facility.

System of Helicopter Emergency Medical Services in Nepal: A Study at Nepal Mediciti Hospital.

OBJECTIVE: Nepalis have benefited from helicopter emergency medical services (HEMS) since 2013. Helicopters are coordinated from private companies for medical transport. There are no helicopters dedicated solely for emergency medical services. Private helicopter companies and hospitals collaborate to transfer patients. Mountainous terrain, traffic infrastructure problems, and distance to rural facilities designate HEMS as the preferred method for transferring patients in Nepal. This article discusses the 2 methods used to fly patients between facilities and from scene calls. The first and preferred method is when patients received medical support from trained personnel en route to the appropriate facility. This method allows for quicker access to a physician with appropriate care throughout a transfer. The second method used occurs when patients were flown with no medical team or trained care onboard the helicopter. Regardless of the method used, HEMS has proven to be beneficial because it limits out-of-hospital time, alleviates patient load from overwhelmed hospitals, and delivers patients to critical care facilities out of reach by ground emergency medical services. The aim of this study was to interpret the current system of helicopter emergency medical services in Nepal and to determine the difference in patient outcomes when transferred with care onboard and without. METHODS: This was a retrospective study of patients who were transferred by helicopter to Mediciti Hospital in Nepal from November 2017 to December 15, 2019. RESULTS: During the study, a total of 425 patients were transferred by helicopter. Two hundred forty-three (57.18%) patients were moved with the support of the medical team onboard. One hundred eighty-two (42.82%) were flown without medical support. Of the 243 patients, 173 (71.19%) were medical, and 70 (28.81%) had suffered traumatic injuries. From the 182 nonsupported patients, 115 (63.19%) were medical, and 67 (36.81%) were traumatic. One hundred eighty (74.07%) of the patients with medical support went to the intensive care unit (ICU). Forty-two (23.07%) without medical support went to the ICU. Ninety-one (50.55%) patients who received support went to the ICU and stayed for 1 to 5 days. Thirty-eight (90.48%) patients with no medical support en route stayed 1 to 5 days in the ICU. Of the 243 patients who received medical support en route, 69 died (28.4%) during the course of treatment in the hospital. Of the 182 who were flown without medical care during transport, 6 (3.33%) died. CONCLUSION: HEMS effectiveness and usefulness are rapidly growing in Nepal. Having trained medics onboard delivering care can be beneficial. When patients are critically ill, it is preferred to fly them via helicopter with a medical crew onboard.

Cancer cell detection by a heart-shaped dual-core photonic crystal fiber sensor.

This paper contributes a novel design of sensor with a heart-shaped dual-core photonic crystal fiber (PCF) to detect cancerous cells in human cervical, blood, adrenal glands, and breast. Cancer-infected cells and their normal cells are considered in liquid form having their own refractive indices. In the designed PCF, the two heart-shaped cores separated by a large circular air hole serve as two independent waveguides. The large circular air hole is infiltrated by sample cells from different body parts. Detection of cancer-contaminated cells by the proposed PCF is based on the mode-coupling theory. According to the mode-coupling theory, the guided optical light transmits periodically from one core to another, throughout the PCF length. During this transmission, the optical light interacts with the cancerous cell, which is filled in the center air hole of the PCF. Due to this interaction, the dip wavelength of the transmission spectrum is sensitive to the corresponding cancerous cell filled in the center air hole of the PCF. The variation in the PCF transmission spectrum for cancerous cells and their normal cells is observed by using the finite element method. The dip wavelength shift of the cancer cell in reference to its normal cell has been measured from the transmission spectrum to determine the sensing performance of the proposed sensor. The sensitivity achieved of the proposed sensor for cervical cancer cell, blood cancer cell, adrenal gland cancer cell, and breast cancer cells are 7916.67 nm/RIU, 8571.43 nm/RIU, 9285.71 nm/RIU, and 10,000 nm/RIU, respectively, with a maximum detection limit of 0.024. Therefore, the proposed PCF sensor suggests high sensitivity with a rapid cancer detection mechanism.

Host-pathogen interactome analysis of Chikungunya virus envelope proteins E1 and E2.

The envelope proteins of Chikungunya virus (CHIKV) are known to play crucial roles in viral infection and spread. Although the role of envelope proteins in viral infection has been studied, the cellular interactors of these proteins are still elusive. In the present study, the ectodomains of CHIKV envelope proteins (E1 and E2) have been used for a high throughput yeast two-hybrid (Y2H) screening to identify the interacting host protein partners. Following a comparative analysis between the viral-host protein interaction data generated from Y2H and computational approach, five host proteins interacting with E1 and three host proteins interacting with E2 common to both datasets were identified. These associations were further verified independently by pull down and protein interaction ELISA. The identified interactions shed light on the possible cellular machinery that CHIKV might be employing during viral entry, trafficking, and evasion of immune system.

Network mapping among the functional domains of Chikungunya virus nonstructural proteins.

Formation of virus specific replicase complex is among the most important steps that determines the fate of viral transcription and replication during Chikungunya virus (CHIKV) infection. In the present study, the authors have computationally generated a 3D structure of CHIKV late replicase complex on the basis of the interactions identified among the domains of CHIKV nonstructural proteins (nsPs) which make up the late replicase complex. The interactions among the domains of CHIKV nsPs were identified using systems such as pull down, protein interaction ELISA, and yeast two-hybrid. The structures of nsPs were generated using I-TASSER and the biological assembly of the replicase complex was determined using ZRANK and RDOCK. A total of 36 interactions among the domains and full length proteins were tested and 12 novel interactions have been identified. These interactions included the homodimerization of nsP1 and nsP4 through their respective C-ter domains; the associations of nsP2 helicase domain and C-ter domain of nsP4 with methyltransferase and membrane binding domains of nsP1; the interaction of nsP2 protease domain with C-ter domain of nsP4; and the interaction of nsP3 macro and alphavirus unique domains with the C-ter domain of nsP1. The novel interactions identified in the current study form a network of organized associations that suggest the spatial arrangement of nsPs in the late replicase complex of CHIKV.

PCF-based Sensors for Biomedical Applications-A Review.

The article provides a comprehensive overview of the current and future advances of Photonic crystal fiber (PCF) based biosensors, the research explores the impact of structural parameter variations on phase matching conditions, by investigating pitch, air hole diameter, and gold layer thickness. Currently, these surface plasmon resonance (SPR) biosensors demonstrate the ability to detect a range of biological substances such as glucose, pH, serum proteins, and similar chemicals. They have the capacity to directly identify bio-components in urine, blood, and saliva, as well as pathogens, bacteria, and contaminants in food, water, and air. The study investigates by presenting the fundamental principles of PCF biosensors, highlighting their comparative benefits over conventional biosensors. Recent studies utilizing PCF biosensors for various application are reviewed, the findings of the review suggest that the integration of SPR enhances the sensing capabilities of these biosensors, making them promising tools for diverse applications in the field of biosensing.

An Interesting Case of Refractory Thrombotic Thrombocytopenic Purpura in the First Trimester of a Twin Pregnancy.

Thrombotic thrombocytopenic purpura (TTP) is a rare pregnancy complication characterized by microangiopathic hemolytic anemia and consumption thrombocytopenia. We herein describe the case report of a 32-year-old woman who was six weeks pregnant with twins and developed thrombotic thrombocytic purpura (TTP). The patient had a history of sickle cell trait, migraines, and preeclampsia. She presented with complaints of nausea, fatigue, sore throat, and cough and was found to be anemic with a hemoglobin of 7 g/dl and thrombocytopenic with a platelet count of 8 x 103/µL. The patient was promptly initiated on steroids and plasmapheresis with an excellent initial response. However, after three days, she developed a sudden onset headache and shortness of breath, and repeat labs showed worsening anemia (7.3 g/dl) and thrombocytopenia (8 x 103/µL). ADAMTS13 activity was significantly low at 2%. Plasmapheresis was continued, and caplacizumab and rituximab treatment was initiated. The fetal ultrasound showed no cardiac activity in the fetal poles, and the patient had a dilation and curettage (D&C) for a missed abortion. She was discharged with a prednisone taper, daily caplacizumab, and weekly rituximab. This case report underscores the criticality of the prompt identification of TTP in its early stages, and appropriate management strategies for patients with refractory TTP (rTTP), including plasmapheresis, caplacizumab, and rituximab.

Au-TiO2 Coated Photonic Crystal Fiber Based SPR Refractometric Sensor for Detection of Cancerous Cells.

A surface plasmon resonance (SPR) refractometric sensor based on gold (Au) and titanium dioxide (TiO2) coated Photonic Crystal Fiber (PCF) is presented for the quick detection of various types of cancerous cells. The cancerous cells and their corresponding normal cells are both considered to be liquid cells each with their unique refractive index (RI). Normally these cells are found in liquid form in the suitable media (food) required to live the cancerous/normal cell lines. Also in our detection case, liquid samples are easy to pump into the sensing channel of the proposed PCF by employing either pressure or capillary forces.The proposed PCF sensor works on the SPR principle, with the Au coating serving as the plasmonic material. This sensor is investigated using the COMSOL Multiphysics software computational tool that is based on the full-vector finite element method (FEM). A TiO2 coating has been applied to enhance adhesion between the Au layer and the PCF surface. Above the Au coating, cancerous cells samples are filled into the PCF. When the core mode of the PCF is coupled with the surface plasmon polariton (SPP) mode under the specific resonance circumstances, SPR will occur on the interface of the gold-sample cell, and in the core mode, the loss peak is observed at the resonance wavelength. Cancerous cells samples have a distinct loss peak than normal cells samples therefore the cancerous cells can be diagnosed by measuring the shift in resonance wavelength corresponding to the loss peak of cancerous and their normal cells samples. The proposed sensor may identify various cancerous cells such as MDAMB-231, MCF-7, PC12, HeLa, and Jurkat for the diagnosis of breast cancer type-1, breast cancer type-2, adrenal glands, cervical, and blood cancer respectively. The computed wavelengths sensitivities of the proposed PCF are 9428.57nm/RIU, 10714.28nm/RIU, 7571.43nm/RIU, 5500nm/RIU, and 6000nm/RIU for the MDAMB-231, MCF-7, PC12, HeLa, and Jurkat cancerous cells, respectively. However, for various cancerous cells, the maximum amplitude sensitivity varies from -1387 RIU-1 to -1599 RIU-1. Moreover, the sensor resolution ranges between 0.93 ×10-5 RIU and 1.82 ×10-5 RIU with a 0.024 maximum detection limit. Because of its improved sensing capability, the presented SPR refractometric sensor is appropriate for the early detection of cancerous cells.

Timing of Surgery and Pre-operative Physiological Parameters as Clinical Predictors of Surgical Outcomes in Traumatic Subaxial Cervical Spine Fractures and Dislocations.

Objective To evaluate the risk factors and outcomes in patients surgically treated for subaxial cervical spine injuries with respect of the timing of surgery and preoperative physiological parameters of the patient. Methods 26 patients with sub-axial cervical spine fractures and dislocations were enrolled. Demographic data of patients, appropriate radiological investigation, and physiological parameters like respiratory rate, blood pressure, heart rate, PaO2 and ASIA impairment scale were documented. They were divided pre-operatively into 2 groups. Group U with patients having abnormal physiological parameters and Group S including patients having physiological parameters within normal range. They were further subdivided into early and late groups according to the timing of surgery as U early , U late, S early and S late . All the patients were called for follow-up at 1, 6 and 12 months. Results 56 percent of patients in Group S had neurological improvement by one ASIA grade and a good outcome irrespective of the timing of surgery. Patients in Group U having unstable physiological parameters and undergoing early surgical intervention had poor outcomes. Conclusion This study concludes that early surgical intervention in physiologically unstable patients had a strong association as a risk factor in the final outcome of the patients in terms of mortality and morbidity. Also, no positive association of improvement in physiologically stable patients with respect to the timing of surgery could be established.

Intraviral protein interactions of Chandipura virus.

Chandipura virus (CHPV) is an emerging rhabdovirus responsible for several outbreaks of fatal encephalitis among children in India. The characteristic structure of the virus is a result of extensive and specific interplay among its five encoded proteins. The revelation of interactions among CHPV proteins can help in gaining insight into viral architecture and pathogenesis. In the current study, we carried out comprehensive yeast two-hybrid (Y2H) analysis to elucidate intraviral protein-protein interactions. All of the interactions identified by Y2H were assessed for reliability by GST pull-down and ELISA. A total of eight interactions were identified among four viral proteins. Five of these interactions are being reported for the first time for CHPV. Among these, the glycoprotein (G)-nucleocapsid (N) interaction could be considered novel, as this has not been reported for any members of the family Rhabdoviridae. This study provides a framework within which the roles of the identified protein interactions can be explored further for understanding the biology of this virus at the molecular level.

Isolated Myeloid Sarcoma: A Diagnostic Dilemma.

Myeloid sarcoma (MS)/granulocytic sarcoma/myeloblastoma/chloroma is a rare extramedullary proliferation of blast cells of one or more myeloid lineages along with the destruction of the normal architecture of adjacent tissue. Isolated MS is a rare entity with an incidence of 0.7 out of 1 million children and 2 out of 1 million adults. Varied clinical presentation, the rarity of the diagnosis, inadequate immunophenotyping, and lack of available literature makes the disease difficult to manage. Here, we report a case of MS in a 44-year-old male with an initial presentation of testicular mass without bone marrow involvement, causing diagnostic challenges. In this case report, we discuss the pathogenesis, diagnostic challenges, and therapeutic options of MS.

The impact of curated educational videos on pathology health literacy for patients with a pancreatic, colorectal, or prostate cancer diagnosis.

Despite patients having increased access to their own electronic health record (EHR) in recent times, patients are often still not considered a primary audience of pathology reports. An alternative to in-person patient education is the use of multimedia programming to enhance health literacy. Curated video presentations designed to explain diagnosis-specific pathology terms were reviewed by a board-certified pathologist and oncologist team and then shown to patients with a primary diagnosis of either pancreatic, colorectal, or prostate cancer in-clinic; these patients then completed a secure electronic survey immediately afterwards. Seventy patients were surveyed, with 91% agreeing or strongly agreeing that the video they watched increased their understanding of the medical terms used in their pathology reports, with a corresponding average Likert score (ALS) of 4.21 (SD = 0.77, CI = ± 0.18). Furthermore, 95% agreed or strongly agreed that the video they watched both enhanced their understanding of the role of the pathologist in diagnosing cancer (ALS = 4.27; SD = 0.65, CI = ± 0.15) and reported they found the video useful (ALS = 4.27; SD = 0.53, CI = ± 0.13). Curated videos such as those utilized in this study have the potential to increase patient health literacy and inform patients of the multidisciplinary nature of cancer diagnosis.

Efficacy and safety of next-generation tick transcriptome-derived direct thrombin inhibitors.

Despite their limitations, unfractionated heparin (UFH) and bivalirudin remain standard-of-care parenteral anticoagulants for percutaneous coronary intervention (PCI). We discovered novel direct thrombin inhibitors (DTIs) from tick salivary transcriptomes and optimised their pharmacologic activity. The most potent, ultravariegin, inhibits thrombin with a Ki of 4.0 pM, 445-fold better than bivalirudin. Unexpectedly, despite their greater antithrombotic effect, variegin/ultravariegin demonstrated less bleeding, achieving a 3-to-7-fold wider therapeutic index in rodent thrombosis and bleeding models. When used in combination with aspirin and ticagrelor in a porcine model, variegin/ultravariegin reduced stent thrombosis compared with antiplatelet therapy alone but achieved a 5-to-7-fold lower bleeding time than UFH/bivalirudin. Moreover, two antibodies screened from a naïve human antibody library effectively reversed the anticoagulant activity of ultravariegin, demonstrating proof-of-principle for antidote reversal. Variegin and ultravariegin are promising translational candidates for next-generation DTIs that may reduce peri-PCI bleeding in the presence of antiplatelet therapy.

Emulsion PCR made easy.

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.

An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation.

PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli.

Virtual screening of inhibitors against Envelope glycoprotein of Chikungunya Virus: a drug repositioning approach.

Chikungunya virus (CHIKV) a re-emerging mosquito-borne alpha virus causes significant distress which is further accentuated in the lack of specific therapeutics or a preventive vaccine, mandating accelerated research for anti-CHIKV therapeutics. In recent years, drug repositioning has gained recognition for the curative interventions for its cost and time efficacy. CHIKV envelope proteins are considered to be the promising targets for drug discovery because of their essential role in viral attachment and entry in the host cells. In the current study, we propose structure-based virtual screening of drug molecule on the crystal structure of mature Chikungunya envelope protein (PDB 3N41) using a library of FDA approved drug molecules. Several cephalosporin drugs docked successfully within two binding sites prepared at E1-E2 interface of CHIKV envelop protein complex with significantly low binding energies. Cefmenoxime, ceforanide, cefotetan, cefonicid sodium and cefpiramide were identified as top leads with a cumulative score of -67.67, -64.90, -63.78, -61.99, and - 61.77, forming electrostatic, hydrogen and hydrophobic bonds within both the binding sites. These shortlisted leads could be potential inhibitors of E1-E2 hetero dimer in CHIKV, hence might disrupt the integrity of envelope glycoprotein leading to loss of its ability to form mature viral particles and gain entry into the host.

Expression, purification and functional characterization of recombinant hypervariable region (HVR) of Chikungunya virus nsP3 protein.

One of the most important rapidly emerging mosquito-borne alphavirus is Chikungunya virus (CHIKV). There is a necessity to develop anti-CHIKV therapeutics, as neither antiviral drug nor vaccines have been licensed yet. Several CHIKV proteins are being studied worldwide, but non-structural protein 3 (nsP3) has been less explored. This protein consists of three domains: macrodomain, alphavirus unique domain (AUD) and hypervariable region (HVR). The proline-rich regions of HVR contain SRC homology 3 (SH3)-binding domain which is essential for its functionality. Interaction of these motifs with host amphiphysin protein is crucial for viral RNA replication. Restricting the interactions of HVR could lead to inhibition of viral life cycle. Therefore, the present study focuses on purification of HVR protein and its structural and functional assay for therapeutic intervention in future use. In order to obtain purified protein, HVR region was amplified from TOPO clones of nsP3 of IND-06-Guj strain and cloned into expression vector. Expression and solubilization of the protein were optimized at various conditions of salt, detergent and imidazole before purification. The soluble recombinant HVR (His-HVR) protein was purified using affinity chromatography. Purified protein was analyzed for structural studies and functional assays. Circular dichroism of His-HVR protein was performed for structural study, and it was observed that it consists of mostly random coils. For functional assay, co-pull down of His-HVR protein was performed with endogenous amphiphysin-I protein of N2a cells and was analyzed using Western blotting. This purified protein obtained could be used as a potential target reagent for novel therapeutic interventions in the future.

BCNU toxicity presenting with a large pericardial and pleural effusion.

BACKGROUND: Carmustine (BCNU), 1, 3-bis (2-chloroethyl)-1-nitosourea, is an alkylating agent which is commonly used as a part of conditioning regimen for autologous peripheral blood stem cell transplantation. Despite the widespread use of BCNU therapy in adults, cardiopulmonary toxicity presenting with cardiac tamponade has not been reported. CASE REPORT: We present a patient who received BCNU as part of her conditioning regimen followed by autologous peripheral blood stem cell transplantation. This patient subsequently developed bilateral upper lobe pulmonary infiltrates with bilateral pleural effusions and a large pericardial effusion which was a result of BCNU toxicity. CONCLUSIONS: Early institution of glucocorticoids for patients receiving high dose BCNU containing chemotherapeutic regimens has shown to significantly reduce BCNU induced cardio-pulmonary toxicity.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.