Monoclonal antibody and protein therapeutic formulations for subcutaneous delivery: high-concentration, low-volume vs. low-concentration, high-volume.

Biologic drugs are used to treat a variety of cancers and chronic diseases. While most of these treatments are administered intravenously by trained healthcare professionals, a noticeable trend has emerged favoring subcutaneous (SC) administration. SC administration of biologics poses several challenges. Biologic drugs often require higher doses for optimal efficacy, surpassing the low volume capacity of traditional SC delivery methods like autoinjectors. Consequently, high concentrations of active ingredients are needed, creating time-consuming formulation obstacles. Alternatives to traditional SC delivery systems are therefore needed to support higher-volume biologic formulations and to reduce development time and other risks associated with high-concentration biologic formulations. Here, we outline key considerations for SC biologic drug formulations and delivery and explore a paradigm shift: the flexibility afforded by low-to-moderate-concentration drugs in high-volume formulations as an alternative to the traditionally difficult approach of high-concentration, low-volume SC formulation delivery.

Hyper-transcription of heat shock factors and heat shock proteins safeguard caprine cardiac cells against heat stress.

The present study was undertaken to document the transcriptional abundance of heat shock factors and heat shock proteins and their role in survivability of caprine cardiac cells during heat stress. Cardiac tissues were collected from different goats (n = 6) and primary cardiac cell culture was done in an atmosphere of 5% CO2 and 95% air at 38.5 °C. Cardiac cells accomplished 70-75% confluence after 72 h of incubation. Confluent cardiac cells were exposed to heat stress at 42 °C for 0 (control), 20, 60, 100 and 200 min. Quantitative RT-PCR for ß2m (internal control), heat shock factors (HSF1, HSF2, HSF4, HSF5), heat shock proteins (HSP10, HSP40), and Caspase-3 was done and their transcriptional abundance was assessed by Pfaffl method. Transcriptional abundance of HSF1, HSF2, and HSF4 did not change at 20 min, increased (P < 0.05) from 60 to 200 min and reached zenith at 200 min of heat exposure. However, transcriptional abundance of HSF5 was gradually escalated (P < 0.05) from 20 to 200 min and registered highest at 200 min of heat exposure. Transcriptional abundance of HSP10 and HSP40 followed an similar pattern like that of HSF5. Transcriptional abundance of Caspase-3 was significantly down-regulated at 200 min of heat exposure. It could be speculated that over-expression of HSFs and HSPs might have reduced Caspase-3 expression at 200 min of heat exposure suggesting their involvement in cardiac cells survival under heat stress. Moreover, hyper-expression of HSFs and HSPs could maintain the integrity and endurance of cardiac tissues of goats under heat stress.

Transcription pattern of key molecular chaperones in heat shocked caprine cardiac fibroblasts.

The present study was attempted to unveil the impact of heat stress on transcription pattern of major heat shock response genes in caprine cardiac fibroblasts. Cardiac tissues (n = 6) were collected and primary cardiac cell culture was done. Cultured cardiac fibroblasts were kept in an atmosphere of 5% CO2 and 95% air at 38.5 °C. Cardiac cells achieved 70-75% confluence after 72 hours of incubation. Heat stress was induced on confluent cardiac fibroblasts at 42 °C for 0 (control), 20, 60, 100 and 200 min. Quantitative RT-PCR for ß2m (internal control), HSP60, HSP70, HSP90, and HSP110 was done and their transcription pattern was assessed by Pfaffl method. HSP60, HSP90, and HSP110 transcription did not differ at 20 min, up-regulated (p < 0.05) from 60 to 200 min and registered highest at 200 min of heat exposure. HSP70 transcription was gradually escalated (p < 0.05) time dependently from 20 to 200 min and reached zenith at 200 min of heat exposure. Differential induction in transcription of key molecular chaperones at various durations of heat exposure might reduce cardiac fibroblasts apoptosis and thus could maintain cardiac tissue function during heat stress.

Gap Statistics for Confined Particles with Power-Law Interactions.

We consider the N particle classical Riesz gas confined in a one-dimensional external harmonic potential with power-law interaction of the form 1/r^{k}, where r is the separation between particles. As special limits it contains several systems such as Dyson's log-gas (k→0^{+}), the Calogero-Moser model (k=2), the 1D one-component plasma (k=-1), and the hard-rod gas (k→∞). Despite its growing importance, only large-N field theory and average density profile are known for general k. In this Letter, we study the fluctuations in the system by looking at the statistics of the gap between successive particles. This quantity is analogous to the well-known level-spacing statistics which is ubiquitous in several branches of physics. We show that the variance goes as N^{-b_{k}} and we find the k dependence of b_{k} via direct Monte Carlo simulations. We provide supporting arguments based on microscopic Hessian calculation and a quadratic field theory approach. We compute the gap distribution and study its system size scaling. Except in the range -1-2 with both Gaussian and non-Gaussian scaling forms.

Active microrheology using pulsed optical tweezers to probe viscoelasticity of lamin A.

Lamins are nucleoskeletal proteins of mammalian cells that stabilize the structure and maintain the rigidity of the nucleus. These type V intermediate filament proteins which are predominantly of A and B types provide necessary tensile strength to the nucleus. Single amino acid missense mutations occurring all over the lamin A protein form a cluster of human diseases termed as laminopathies, most of which principally affect the muscle and cardiac tissues responsible for load bearing functionalities of the body. One such mutation is A350P which causes dilated cardiomyopathy in patients. It is postulated that a change from alanine to proline in the α-helical coiled-coil forming 2B rod domain of the protein might severely disrupt the propensity of the filaments to polymerise into functional higher order structures required to form a fully functional lamina with its characteristic elasticity. In this study, we have elucidated for the very first time, the application of active microrheology employing oscillating optical tweezers to investigate any alterations in the viscoelastic parameters of the mutant protein meshwork in vitro, which might translate into possible changes in nuclear plasticity. We confirmed our findings from this robust yet fast method by imaging both the wild type and mutant lamin A networks using a super resolution microscope, and observed changes in the mesh size which corroborate our measured changes in the viscoelastic parameters of the lamins. This method could thus be extended to conduct microrheological measurements on any intermediate filament protein thus bearing significant implications in laminopathies and other diseases associated with intermediate filaments.

Origin, genetic diversity and evolution of Andaman local duck, a native duck germplasm of an insular region of India.

Domestic ducks are of paramount importance as a cheap source of protein in rural India. Andaman local duck (ALD) is an indigenous avian genetic resource of Andaman and Nicobar islands (ANI) and is mainly distributed in Middle and Northern parts of these islands. Negligence has brought this breed on the edge of extinction necessitating immediate conservation efforts. Here, we report the genetic diversity, population structure and matrilineal genetic root of ALD. Partial mtDNA D-loop sequences were analyzed in 71 ALD samples and analysis revealed 19 polymorphic sites and 13 haplotypes. Estimated haplotype (Hd ± SD) and nucleotide diversity (π ± SD) were 0.881 ± 0.017 and 0.00897 ± 0.00078 respectively. The high genetic diversity of ALD indicates introgression of genetic material from other local duck breeds. In addition, it can be postulated that ALD bearing high genetic diversity has strong ability to adapt to environmental changes and can withstand impending climate change. Phylogenetic and network analysis indicate that ALD falls under Eurasian clade of mallard and ALD forms three clusters; one cluster is phylogenetically close to Southeast Asian countries, one close to Southern part of mainland India and the third one forms an independent cluster. Therefore, ALD might have migrated either from Southeast Asian countries which enjoy a close cultural bondage with ANI from time immemorial or from Southern part of India. The independent cluster may have evolved locally in these islands and natural selection pressure imposed by environmental conditions might be the driving force for evaluation of these duck haplotypes; which mimics Darwin's theory of natural selection. The results of the study will be beneficial for formulating future breeding programme and conservation strategy towards sustainable development of the duck breed.

Role of seminal MDA, ROS, and antioxidants in cryopreservation and their kinetics under the influence of ejaculatory abstinence in bovine semen.

Despite many cryopreservation techniques in bovine semen, various stressors' detrimental effects remain a significant issue. The present study targeted to assess the role of semen quality parameters, sperm function tests, lipid peroxidation, reactive oxygen species (ROS), and different antioxidants in the cryopreservation of bovine semen. Further, the kinetics of lipid peroxidation, ROS, and antioxidants on repeated semen collection under short ejaculatory abstinence were studied. We designed a comparative study where bulls were grouped into good and low freezable semen groups (Freeze-groups) based on their post-thaw motility. All the bulls included had similar initial motility and qualified minimum standards for initial semen parameters viz. semen volume and sperm concentration. The present study detected a higher lipid peroxidation and ROS viz. superoxide anions (•O2-) and a lower total antioxidant capacity (TAC) in the low freeze-group compared to the good freeze-group. The ROS and antioxidants showed unique kinetics on repeated semen collection at short intervals, and no significant change was detected in semen volume, sperm motility, and sperm concentration. This study detected higher head abnormalities and poor acrosome integrity in the low freeze-groups. The present study results indicated that the sperm head might be the most vulnerable part of the sperm to cryopreservation stress. The present study finds significantly higher lipid peroxidation and ROS levels and reduced antioxidant capacity as the primary reasons for low cryopreservability. Further, repeated semen collection with a shorter or lack of abstinence does not impose any significant change in the semen volume and sperm concentration; moreover, it could be beneficial for higher antioxidant levels and lower lipid peroxidation levels. As seminal plasma has both inhibitory and stimulatory roles in sperm function and cryopreservation, identifying the critical role players of seminal plasma and identifying sperm related changes in cryopreservation could predict the cryopreservability potential of semen.

Impact of thermal stress on expression dynamics of HSP60 in cardiac fibroblast cells of goat.

The present study was aimed to determine the impact of thermal stress on expression dynamics of heat shock protein 60 (HSP60) mRNA in cultured cardiac fibroblast cells of the goat. The heart tissues (n = 6) from different goats were used for the culture study. The cardiac fibroblast cells were cultured and subjected to thermal stress at 42 °C for 0, 20, 60 and 100 min. The relative abundance of HSP60 mRNA was assessed by quantitative real-time PCR (qRT-PCR). The cardiac cells exposed to thermal stress at 42 °C for 0 min was taken as control. The relative abundance of HSP60 mRNA did not change at 20 min of thermal stress as compared to control. Thereafter, the relative abundance of HSP60 mRNA was significantly up-regulated (p < 0.05) at 60 min and 100 min of thermal stress. However, the highest mRNA expression of HSP60 was noticed at 100 min of thermal stress. The present study indicates that, thermal stress modulates the mRNA expression HSP60 in cultured caprine cardiac fibroblast cells.

Synthesis of mesoscale ordered two-dimensional π-conjugated polymers with semiconducting properties.

Two-dimensional materials with high charge carrier mobility and tunable band gaps have attracted intense research effort for their potential use in nanoelectronics. Two-dimensional π-conjugated polymers constitute a promising subclass because the band structure can be manipulated by varying the molecular building blocks while preserving key features such as Dirac cones and high charge mobility. The major barriers to the application of two-dimensional π-conjugated polymers have been the small domain size and high defect density attained in the syntheses explored so far. Here, we demonstrate the fabrication of mesoscale ordered two-dimensional π-conjugated polymer kagome lattices with semiconducting properties, Dirac cone structures and flat bands on Au(111). This material has been obtained by combining a rigid azatriangulene precursor and a hot dosing approach, which favours molecular diffusion and eliminates voids in the network. These results open opportunities for the synthesis of two-dimensional π-conjugated polymer Dirac cone materials and their integration into devices.

Tracing the genetic root of Trinket cattle: an endangered cattle population of a small isolated island of Nicobar group of islands.

Trinket cattle are the inhabitant of a small island called Trinket, which is one of the picturesque islands of Andaman. This herd is thought to be of Danish leftover during their dynasty in Nicobar archipelago. When the island was abandoned by foreign invaders, indigenes utilized the animals for the purpose of meat. As a result, the cattle became semi-feral in nature. After the Great Sumatra earthquake and tsunami of Indian Ocean in 2004, Trinket island was left abandoned by indigenes and the cattle became totally feral in nature. To trace the genetic root of the cattle, this study has been undertaken based on the sequence information of the mitochondrial D-loop and cytochrome b gene. The genomic DNA was extracted from the blood samples of the Trinket cattle and was used for amplification of mitochondrial markers, and the sequence information was generated by Sanger sequencing. The analysis of sequence information revealed that the Trinket cattle belongs to Bos indicus (I) haplotype, sub-haplotype I2. The presence of I2 sub-haplotype in Trinket cattle may be due to the expansion of this I2 haplotype towards Southeast Asian countries. This is a novel input for the formulation of breeding strategy towards conservation of eco-friendly sustainable livestock in the isolated island ecosystem.

Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers.

For proteome analyses, the tissue samples are mostly preserved either snap frozen or formalin-fixed, paraffin-embedded form. Use of RNAlater-a non-toxic solution primarily used to stabilize the RNA content of samples-in tissue preservation for proteome analysis recently described equally reliable with snap-frozen preservation in human tissues. Even though RNALater storage has great potential in the preservation of Peripheral Blood Mononuclear Cells (PBMC), its impact on the results of proteome analysis is poorly described at qualitative and quantitative measures. The present study investigated protein profiles of RNAlater preserved and fresh PBMCs using three extraction buffers viz. Triton X-100, RIPA and SDS. Proteins are separated in SDS-PAGE and quantified using densitometry. On an average 19.3 bands from fresh and 15.6 bands from RNAlater storage cells were obtained with a molecular weight ranging from 25 to > 250 kDa. RNAlater storage generated a fewer number and lesser quantity of low molecular weight proteins while yielded a similar or high quantity of high molecular weight protein fractions. The principal component analysis showed that Triton X-100 is inferior as compared to SDS and RIPA with respect to their protein bands and quantity yielded. While RNAlater is effective in preserving PBMC for proteome analysis, our findings warrant caution in its use in proteomics experiments especially if the target is low molecular weight proteins.

Impact of heat stress on transcriptional abundance of HSP70 in cardiac cells of goat.

The present study was aimed to document the effect of heat stress on the transcriptional abundance of heat shock protein 70 (HSP70) mRNA in cultured cardiac cells of goat. The heart tissues (n = 6) from different goats were used for the culture study. The cardiac cells obtained from different heart tissues were cultured in 24 well cell culture plates and incubated in a humidified CO2 (5%) incubator at 37 °C. The cardiac cells were allowed to become 75-80% confluent after 72 h of incubation. Thereafter, the cardiac cells were subjected to heat exposure at 42 °C (heat exposed) for 0, 20, 60 and 100 min. The cardiac cells exposed to heat stress at 42 °C for 0 min was taken as control. The relative abundance of HSP70 mRNA was gradually up-regulated (p < .05) from 20 to 100 min of heat exposure and reached the zenith (p < .05) at 100 min of heat challenge. The present finding highlights that, HSP70 could possibly act as a cytoprotective factor and may promote cardiac cell survival against the detrimental effect of heat stress. Moreover, this study may serve as the harbinger to conduct further research work on expression kinetics of HSP70 in cardiac cells of goat including other livestock species.

Complete mitogenome sequencing of Andaman buffalo: an endangered germplasm of Andaman and Nicobar Islands, India.

Andaman buffalo is an indigenous buffalo of Andaman and Nicobar Islands, India. Over the last decade, it has witnessed a rapid decline in population, necessitating its immediate characterization and conservation. The present study reports the complete mitogenome profile of Andaman buffalo which is 16,359 bp in length and comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs and two ribosomal RNAs. In addition, one A + T rich region (D-loop) was also present. A biasness towards A and T base was observed in all the genes. All the PCGs except ND6 were present on heavy strand. Start codons for all the 13 PCGs were ATN codon and abbreviated/truncated stop codons were observed in ND1, ND2, COX3, ND3 and ND4. The phylogenetic analysis revealed that the Andaman buffalo is closely related to buffalo from India and China. The results from this study will help in sketching the conservation plan of the threatened breed.

Harmonically Confined Particles with Long-Range Repulsive Interactions.

We study an interacting system of N classical particles on a line at thermal equilibrium. The particles are confined by a harmonic trap and repel each other via pairwise interaction potential that behaves as a power law ∝∑[under i≠j][over N]|x_{i}-x_{j}|^{-k} (with k>-2) of their mutual distance. This is a generalization of the well-known cases of the one-component plasma (k=-1), Dyson's log gas (k→0^{+}), and the Calogero-Moser model (k=2). Because of the competition between harmonic confinement and pairwise repulsion, the particles spread over a finite region of space for all k>-2. We compute exactly the average density profile for large N for all k>-2 and show that while it is independent of temperature for sufficiently low temperature, it has a rich and nontrivial dependence on k with distinct behavior for -21 and k=1.

Mitogenome analysis of Indian isolate of Rhipicephalus microplus clade A sensu (): A first report from Maritime South-East Asia.

This communication reports a comprehensive profile of mitogenome analysis of Rhipicephalus microplus, isolated and identified from Andaman and Nicobar islands, a part of Maritime South East Asia. Complete mitogenome of Indian isolate of R. microplus (MK234703) was 14,903 bp. Mitochondrial (mt.) genome had 13 protein coding genes (PCGs), 22 tRNAs, two ribosomal subunits and two control regions. All PCGs were located on the H-strand except nad1, nad5, nad4 and nad4L. All start codons were ATN codon and abbreviated stop codons were seen in cox-2-3, nad-5 and cytb. A purine rich tick-box motif has been identified. A tandem repeat unit (TTTATT), described as a region alike to nad1 was identified in 130 bp insertion in between nad1 and tRNA-Glu and in nad1 sequence. Presence of two control regions (CRs) proved that, two CRs have evolved in concert rather than independently. Strong biasness towards A and T in Indian isolate of R. microplus is a typical feature for most of the arthropods. Subtracted values of dn and ds suggested that, there was least effect of nt. sequence of cox1 gene when Indian isolate was compared with other isolates of Rhipicephalus. On the basis of phylogenetic analysis, species of the genus Rhipicephalus could be clustered in three groups; ticks of the genera belonging to sub-family Rhipicephalinae could be grouped in a single cluster. Finally, cox1 sequence of MK234703 indicated that the isolate belonged to clade A sensu Burger et al., 2014 which has not been reported earlier from India.

GhSTOP1, a C2H2 type zinc finger transcription factor is essential for aluminum and proton stress tolerance and lateral root initiation in cotton.

Aluminum (Al) and proton (H+ ) ions are major acid soil stress factors deleteriously affecting plant root growth and crop yield. In our preliminary studies, cotton (Gossypium hirsutum L.) seedlings displayed very sensitive phenotypes to Al and H+ rhizotoxicities. Based on previous Arabidopsis results, we aimed to characterise the Al stress responsive Sensitive to Proton rhizotoxicity 1 (GhSTOP1) transcription system in cotton using RNAi-mediated down-regulation. With the help of seed embryo apex explants, we developed transgenic cotton plants overexpressing a GhSTOP1-RNAi cassette with NPTII selection. Kanamycin-tolerant T1 seedlings were further considered for Al and H+ stress tolerance studies. Down-regulation of the GhSTOP1 increased sensitivity to Al and proton rhizotoxicities, and root growth was significantly reduced in RNAi lines. The expression profile of GhALMT1 (Aluminum-activated Malate Transporter 1), GhMATE (Multidrug and Toxic Compound Extrusion), GhALS3 (Aluminium Sensitive 3) and key genes involved in the GABA shunt were down-regulated in the transgenic RNAi lines. Additionally, the lateral root initiation process was delayed and expression of GhNAC1, which is involved in lateral roots, was also suppressed in transgenic lines. Besides, overexpression of GhSTOP1 in Arabidopsis accelerated root growth and AtMATE and AtALMT1 expression under Al stress conditions. These analyses indicate that GhSTOP1 is essential for the expression of several genes which are necessary for acid soil tolerance mechanisms and lateral root initiation.

Chlorogenic acid-mediated chemical defence of plants against insect herbivores.

Chlorogenic acid is one of the most abundant beneficial polyphenols in plants and is well known as a nutritional antioxidant in plant-based foods. Apart from its dietary antioxidant activity, it has been proved to be an efficient defence molecule against a broad range of insect herbivores. In the last two decades, several reports have shown the effectiveness of chlorogenic acid in insect growth deterrence. The pathway for chlorogenic acid biosynthesis in plants was previously elucidated, and metabolic engineering of the principal pathway showed high chlorogenic acid production in tomato plants. Herbivore-mediated induction of chlorogenic acid biosynthesis was also demonstrated both at metabolite and transcript level, although herbivore-mediated molecular regulation of chlorogenic acid biosynthesis is not yet fully elucidated. In this communication, we present our views on the efficacy of chlorogenic acid as an anti-herbivore defence molecule in plants and also discuss its future outlook.

Mitochondrial landscape of indigenous pig germplasm of Andaman and Nicobar Islands.

Nicobari pig and Andaman Desi pig are indigenous pig germplasm of Andaman and Nicobar islands, India. Over the last two decades, the pig breeds witnessed a rapid decline in population, necessitating immediate characterization and conservation. The present study depicts the complete mitochondrial genome sequence of Nicobari pig and Andaman Desi pig. The mitogenomes of both the breeds encode 37 genes including 13 protein coding genes, 22 tRNAs, and two ribosomal RNA genes. In addition, a control region (D-loop) was also present. Phylogenetic analysis showed that Nicobari is phylogenetically close to Banna mini and Breed I pig, whereas Andaman Desi pig is close to Mong cai and Jinhua pig breeds. The results of the study will be helpful for formulating of conservation strategy of the native swine breeds.

Effect of Asymmetric, Charge Balanced Stimuli on Elicited Compound Neural Action Potentials.

Whether via cuff or intrafascicular electrode, peripheral neural stimulations often rely on symmetric, charge balanced paradigms. To date, few investigations have been carried out which systematically decompose the features of a stimulus waveform. Factors such as pulse-width, amplitude, and the timing with which they are presented may have significant effects on the quality of the stimuli. This work seeks to fill this gap in knowledge and share insight into how selection of electrical stimuli may affect the resultant neural activation in peripheral nerves. In particular, we found that, although there is some variance, over the parameter range tested there was not a significant effect on neural fiber recruitment percent due to waveform selection.

Design and assessment of stimulation parameters for a novel peripheral nerve interface.

Bi-directional interfaces for peripheral nerve stimulation and recording aim to improve control and acceptance of sensorized prosthetic limbs. The implantable multimodal peripheral recording and stimulation system (IMPRESS) is an intraneural interface technology supporting a high-density transverse intrafascicular multichannel electrode (hd-TIME). Herein we report on in vivo selectivity studies using a passive hd-TIME, and computational modeling towards optimal stimulation parameters for fiber recruitment.