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The study evaluated deltamethrin resistance in Rhipicephalus sanguineus and Rhipicephalus (Boophilus) microplus tick populations of Kerala in India, using larval packet test (LPT). Dose response data were analysed by the probit method, the LC50 and LC95 of deltamethrin against ticks were determined by applying regression equation analysis to the probit-transformed data of mortality. In R. sanguineus, 50 per cent of isolates were found resistant at discriminating dose (600 ppm) by larval packet test. The p value obtained upon regression analysis was < 0.05 and was considered as significant. A majority of R. (B.) microplus were found to be susceptible to deltamethrin. However, these susceptible isolates survived doses which were twice the recommended doses (1.25 ppm – 100 ppm). The p value of isolates except isolate 1 and 5 were < 0.05 and statistically significant. The results highlight acaricide resistance to be one of the reasons for the alarming prevalence of tick-borne haemoparasites in Kerala and demand urgent interventions to ameliorate the resistance by alternate control strategies.

A total of 80 snails collected from three habitats viz., uncultivated paddy fields, permanent water bodies and catchment area of dams in Palakkad district of Kerala were screened for presence of echinostome infection. The collected snails were identified morphologically as Indoplanorbis exustus and Lymnaea luteola. Indoplanorbis exustus was found to be the most predominant snail species followed by L. luteola with a prevalence rate of 57.5 and 42.5 per cent, respectively. The prevalence rate of snails was higher in uncultivated paddy fields followed by permanent water bodies and catchment area of dams. Overall prevalence of echinostome infection in snails was found to be 11.2 per cent. Prevalence of infection in I. exustus constituted 13 per cent while that in L. luteola was nine per cent. Dissection of positive snails revealed intra molluscan stages like sporocyst.

The incidence of lungworms (Metastrongylus salmi) in domesticated pigs has been reported. The male and female lungworms were identified by morphological features. Histopathology of the lungs revealed marked lymphoid hyperplasia in the peribronchiolar area. Interstitium showed marked lymphoid reaction, thickening of alveolar walls with presence of congestion, haemorrhage and hemosiderin pigment deposits. Neovascularisation, emphysema and syncytia formation in the interstitium were also observed. Cut-section of the parasite could be observed in the bronchiolar lumen and hyperplasia of the bronchiolar epithelium could be observed. The bronchiolar wall was infiltrated with mononuclear cells and hyperplasia of the Bronchiole Associated Lymphoid Tissue (BALT) was significant. [ABSTRACT FROM AUTHOR]

The article describes the culture of microfilaria of Brugia spp. for the extraction of excretory-secretory protein. The microfilariae were separated from the various components of canine blood using HiSep™ LSM by gradient centrifugation method. The microfilariae were cultured in RPMI-1640 media and incubated at 37°C. The spent media was collected at 12 hour intervals, filtered, pooled and stored at -50°C. Protein estimation was done by Bradford’s method. The excretory-secretory protein produced can further be used in diagnostic tests.

To study the reproductive biology of Horabagrus brachysoma (Günther, 1864), monthly samples were collected from the riverine stretches of Lake Vembanad during 2001-2002. The sexes can be distinguished only during the breeding season; male to female ratio in the exploited catch was 1 : 1.97. The mean L T at first maturity ( L 50) was 18.5 cm in females and 17.5 cm in males. Absolute fecundity varied between 1100 and 124 000, with a mean of 20 472. The ovary showed group synchronous development and the oocyte diameter varied between 0.13 and 1.63 mm. The relatively high fecundity and hardy nature of H. brachysoma may favour the expansion of this indigenous catfish throughout the culture systems. [ABSTRACT FROM AUTHOR]

Aim: Canine babesiosis is an important vector-borne hemoparasitic disease caused by Babesia canis vogeli and Babesia gibsoni, in India. The communication places on record the salient findings of the study directed to detect and characterize the pathogenic B. gibsoni isolates of Kerala state. Materials and Methods: A total of 150 dogs were examined for the presence of hemoparasites by light microscopy as well as by PCR targeting the 18S rRNA gene of B. gibsoni. Hematological parameters were also analysed. Phylogenetic tree was constructed based on Tamura kei model adopting ML method. Results: A sensitive and specific polymerase chain reaction assay was developed with newly designed primer pair BAGI-F/ BAGI-R for the amplification of 488 bp fragment of 18S rRNA gene of B. gibsoni. Out of the 150 dogs examined, molecular evidence of B. gibsoni was recorded in 47.3% animals, while light microscopy detected the infection in 26.67% cases. The phylogenetic analyses revealed that B. gibsoni, Kerala, isolate was closest and occurred together with Bareilly isolate. Anemia and thrombocytopenia were the significant hematological alterations in chronic B. gibsoni infection. Conclusion: A high prevalence of natural infection of B. gibsoni was detected among the study population. The affected animals showed anaemia and thrombocytopenia. Phylogenetic analysis of this pathogenic isolate from south India revealed the closest similarity with Bareilly isolates.

Poultry farming is the most organized and economically viable sector in animal agriculture. As sub-lethal AF is a constant threat to broiler industry and produces immunosuppression, birds would be more prone to coccidiosis even under reduced infective dose. So it is important to study the response of sub-infectious dose of coccidia in an immunecompromised state. The susceptibility of broiler chicks to sub-infective dose of sporulated oocyst of Eimeria tenella in sub-lethal aflatoxicosis was revealed by significant alterations in serological parameters.

Nematocidal properties of spore crystal mixtures of six Bacillus thuringiensis (Bt) strains (KAU 49, 50, 52, 61, 99 and 424) collected from Western Ghats, a biodiversity hot spot of India, were analysed against Haemonchus contortus larvae isolated from goats. One dose nematocidal assay dose response to lyophilised spore-crystal mixtures (SCM) of the six Bt strains were determined by adding 200 μg/mL of each SCMs to culture plate wells containing aqueous suspension of H. contortus larvae. Out of the strains screened, KAU 50 and 424 were found to possess nematocidal properties. Maximum nematocidal properties were exhibited 7 days post-inoculation of the lyophilised SCMs. The 50 per cent lethal concentrations deduced by log probit analysis for KAU 50 was found to be 130.59 μg/mL, whereas that of KAU 424 was found to be 144.536 μg/mL at 95 per cent confidence level. This is the first report on the nematocidal propery of Bt strains against Haemonchus contortus larvae isolated from goats. Further studies are needed for identification and characterisation of the toxin.

Bivitellobilharzia nairi is a poorly understood trematode belonging to the family Schistosomatidae that has a profound impact on its host species, the elephant. The pernicious effects of the different life stages of this parasite can often be fatal. The first reports of schistosomosis in African and Asian elephants were published by Vogel and Minning (1940) and Mudaliar and Ramanujachari (1945), respectively. The morphologies of ova and adult helminths of B.nairi, the schistosome affecting Indian elephants (Elephas maximus indicus) have been described in detail by Rao and Hiregaudar (1935), Sundaram et al. (1972), Chandrasekharan (1989), Islam (1994) and Vimalraj et al. (2012). According to Bhoyar et al. (2014), the infection remains subclinical in most cases and is only discovered upon post-mortem examination. More recently, Devkota(2015) found that Bivitellobilharzia spp. infects both, captive and wild Asian elephants. Karawita et al. (2015) claimed to report the first death of an Asian elephant due to caecocolic intussusception on account of schistosomosis

Oestrus ovis is a larviparous protelean fly whose larvae parasitize the nasal cavities and adjoining sinuses of sheep and goats. It is primarily a widespread infestation of sheep and rarely occurs in goat, camel, deer, reindeer, elk and ibex (Lapage, 1956; Das and Bhatia,1994). The larvae elicit clinical signs of cavitary myiasis seen as seromucous or mucopurulent nasal discharge, frequent sneezing, incoordination and dyspnoea (Madhu et al., 2014). The migratory larvae may also penetrate and erode dorsal turbinate bones, frontal sinuses and occasionally the skull bones thereby entering into the cerebral cavity, causing false gid (Taylor et al., 2007). In India prevalence of O. ovis has been reported in several places (Chhabra and Ruprah, 1976 ; Jagannath et al., 1989 and Allaie et al., 2016). Myiasis has medical and public health significance if the incidental host is man (Sreejith et al., 2010). The present paper describes the occurrence of nasal myiasis from crossbred Malabari goats for the first time in Kerala and its successful medical management.

Use of phytopesticides has many advantages over conventional chemical insecticides and is receiving worldwide attention as an alternative means of pest control. Preliminary studies were conducted to assess the lousicidal activity of alcoholic extracts of Allium sativum bulbs. In vitro study was carried out on the goat louse, Damalinia caprae using filter paper method. It was found that these crude extracts caused cent percent mortality of adult lice at a concentration of 100 mg/ml at 32 h post- exposure whereas at 50 mg/ml, the same mortality was observed at 48 h postexposure. It is opined that further purification studies and in vivo trials are needed to ascertain the active ingredients responsible for the insecticidal properties of garlic juice extracts.

Mesenteric worm count technique was employed to study the prevalence of intestinal schistosomosis among cattle and buffaloes slaughtered in Thrissur. Of the forty eight mesentery samples processed, 45.4 percent animals carried Schistosoma spindale infections with a significant proportion harbouring moderate infections. Long term chronic infections cause significant production loss to the herd. Prevalence of the disease was very high when compared to the existing reports based on faecal egg detection techniques. The study emphasises the need for developing alternate field level diagnostic tests for schistosomosis.

Concurrent infections of pigeon malaria and avian malaria were detected in pigeons by blood smear examination. The affected birds exhibited symptoms of droopiness, anorexia, weight loss, anaemia and torticollis. Pigeon fly infestation due to Pseudolynchia canariensis, was also noticed in the lofts. Postmortem examination of a dead bird revealed congestion of liver, kidney and intestine along with hepatomegaly and splenomegaly. Affected birds were treated with parenteral administration of Chloroquine which resulted in complete recovery and clinical cure. The present communication reports a concurrent outbreak of Fig. Schizonts of Plasmodium sp. and gamonts of Haemoproteus columbae in the erythrocytes (arrow) Haemoproteus sp. and Plasmodium sp. infection in pigeons and its successful control.

Ornithonyssus bursa, the "tropical fowl mite" is an important acarine parasite that attacks poultry and occasionally man. Ornithonyssus bursa cause anaemia and considerable injury to the skin and is reported for the first time from Kerala. Its morphology, morphometry and treatment using deltamethrin are described.

Schistosomosis in animals due to Schistosoma spindale significantly burdens India's livestock economy because of high prevalence and morbidity and is mostly underdiagnosed from the lack of sensitive tools for field-level detection. This study aimed to clone, express the 22.6-kDa tegument protein of S. spindale (rSs22.6kDa) and to utilise it in a dot enzyme-linked immunosorbent assay for serodiagnosis. RNA was extracted from adult worms recovered from the mesenteries of slaughtered cattle to amplify the gene encoding the 22.6-kDa protein. In silico analysis revealed the protein's secondary structure, consisting of 190 amino acids forming alpha helices (47.89%), extended strands (17.37%), beta turns (8.95%), and random coils (25.79%), with α helices and β sheets in the tertiary structure. Two conserved domains were noted: an EF-hand domain at the N-terminus and a dynein light-chain domain at the C-terminus. Phylogenetic studies positioned the S. spindale sequence as a sister clade to Schistosoma haematobium and Schistosoma bovis. The gene was cloned into a pJET vector and transformed into Escherichia coli Top 10 cells, with expression achieved using a pET28b vector, BL21 E. coli cells, and induction with 0.6 mM isopropyl-β-d-thiogalactopyranoside. The protein's soluble fraction was purified using nickel-chelating affinity chromatography, confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, identifying a distinct immunodominant 22.6-kDa protein. The diagnostic utility was validated using a dot enzyme-linked immunosorbent assay which demonstrated a of sensitivity of 89.47% and specificity of 100%. The study records for the first time the prokaryotic expression and evaluation of the 22.6-kDa tegumental protein of S. spindale , highlighting its potential as a diagnostic antigen for seroprevalence studies in bovine intestinal schistosomosis.

The KRAS gene plays a pivotal role in numerous cancers by encoding a GTPase that upon association with the plasma membrane activates the MAPK pathway, promoting cellular proliferation. In our study, we investigated small molecules that disrupt KRAS's membrane interaction, hypothesizing that such disruption could in turn inhibit mutant RAS signaling. Native mass spectrometry screening of KRAS-FMe identified compounds with a preference for interacting with the hypervariable region (HVR), and surface plasmon resonance (SPR) further refined our selection to graveoline as a compound exhibiting preferential HVR binding. Subsequent nuclear magnetic resonance (NMR) analysis showed that graveoline's interaction with KRAS depends on C-terminal O-methylation. Moreover, our findings revealed multiple interaction sites, suggesting weak engagement with the KRAS G domain. Using nanodiscs as a membrane mimetic, further characterization through NMR and Förster resonance energy transfer (FRET) studies demonstrated graveoline's ability to perturb KRAS membrane interaction in a biochemical setting. Our biophysical approach sheds light on the intricate molecular mechanisms underlying KRAS-ligand interactions, providing valuable insights into understanding the KRAS-associated pathophysiology. These findings contribute to the translational aspect of our study, offering potential avenues for further research targeting KRAS membrane association with the potential to lead to a new class of RAS therapeutics., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

The bidirectional long-distance transport of organelles is crucial for cell body-synapse communication. However, the mechanisms by which this transport is modulated for synapse formation, maintenance, and plasticity are not fully understood. Here, we demonstrate through quantitative analyses that maintaining sensory neuron-motor neuron synapses in the Aplysia gill-siphon withdrawal reflex is linked to a sustained reduction in the retrograde transport of lysosomal vesicles in sensory neurons. Interestingly, while mitochondrial transport in the anterograde direction increases within 12 hours of synapse formation, the reduction in lysosomal vesicle retrograde transport appears three days after synapse formation. Moreover, we find that formation of new synapses during learning induced by neuromodulatory neurotransmitter serotonin further reduces lysosomal vesicle transport within 24 hours, whereas mitochondrial transport increases in the anterograde direction within one hour of exposure. Pharmacological inhibition of several signaling pathways pinpoints PKA as a key regulator of retrograde transport of lysosomal vesicles during synapse maintenance. These results demonstrate that synapse formation leads to organelle-specific and direction specific enduring changes in long-distance transport, offering insights into the mechanisms underlying synapse maintenance and plasticity., Competing Interests: CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interests.

Purpose: Genotyping of Rhipicephalus (Boophilus) microplus for polymorphisms in deltamethrin-resistant loci of sodium channel gene by allele-specific PCR (AS-PCR)., Methods: Adult R. (B.) microplus ticks were collected from naturally infested cattle in Kerala. The larval packet test (LPT) was performed with deltamethrin and dose response data were analysed by probit method. Adult and larval tick DNA were amplified by PCR and later sequenced to identify mutations, if any, in the domain II S4-5 linker and domain III S6 regions in para voltage-gated sodium channel gene, at loci that were previously documented to be associated with deltamethrin resistance. Allele-specific PCR was performed for the amplification of target gene locus (C190A and T2134C) to genotype 1000 larvae each, at these loci. Genotype frequency was statistically analysed by Chi-square test., Results: Bioassay using LPT revealed that LC 50 and LC 95 values of all the R. (B.) microplus isolates in this study were more than double the reported values of reference susceptible strain. Sequence analysis of the PCR amplicons of domain II S4-5 linker of voltage-gated sodium channel gene revealed C190A mutation, A271G mutation as well as A-G mutation at 217th position. AS-PCR done to genotype C190A mutations revealed a frequency of 6%, 15% and 64%, respectively for homozygous-susceptible (SS), heterozygous (RS) and homozygous-resistant (RR) genotypes. In domain III S6 region of the gene, C2121T and A2102T mutations were observed. AS-PCR to genotype the previously reported T2134C mutation revealed 100% SS genotype in R. (B.) microplus isolates of Kerala., Conclusions: Genotyping of R. (B.) microplus isolates of Kerala for target site mutations reportedly associated with deltamethrin resistance revealed a significantly high frequency of resistant genotypes at II S4-5 linker of voltage-gated sodium channel gene. This study forms the first report of such mutations in Kerala, south India and demands serious attention in the light of increased prevalence of tick-borne haemoparasitic infection., (© 2021. Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Brown dog tick, Rhipicephalus sanguineus sensu lato, an important ectoparasite transmitting several pathogens, is the most common tick species infesting dogs. Control of ticks being central to the control of fatal tick-borne diseases, this study attempted to assess the susceptibility/resistance of brown dog ticks to synthetic pyrethroids, the commonly used acaricides against ticks. Larval packet assay revealed 60% of isolates tested to be resistant and tolerant to deltamethrin as per the resistance factor that ranged from 1 to 53.7. Sequence analysis of the PCR amplified product of domain II S4-5 linker of sodium channel gene in R. sanguineus revealed novel polymorphisms, viz., C190A, G215T and T270C. In domain III S6 region of the gene, a T2134C mutation was observed. Genotyping with allele-specific PCR targeting domain II S4-5 linker region using single larvae revealed that most R. sanguineus larvae in the study population were homozygous resistant (RR) genotypes, followed by heterozygous (RS) and homozygous susceptible (SS) genotypes. A higher proportion of RS genotypes was also observed in domain III S6 region. This first report of genotyping of Indian R. sanguineus to analyse synthetic pyrethroid resistance highlights the need to devise alternate control strategies to reduce the brown dog tick population.

The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

The trafficking chaperone PDE6D (also referred to as PDEδ) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a "chemical spring" into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block stemness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future., Competing Interests: The authors declare the following competing financial interest(s): We have filed a patent around second generation inhibitor Deltaflexin-2., (Copyright © 2019 American Chemical Society.)

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6-δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization., (Copyright © 2019 Biophysical Society. All rights reserved.)

The Ras-extracellular signal-regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor-mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress., (Published by Elsevier Inc.)

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects., Competing Interests: The authors declare no conflict of interest.

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell-surface receptors/tyrosine kinases. EGFR- and HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683-696). We have created photostable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/ receptors.

Water quality of four open wells representing four localities around the Kerala Minerals and Metals Ltd industrial area, Chavara, Quilon district was studied fora period of six months from December, 2006 to May 2007 to assess the suitability of the well waters for domestic purposes. The well waters exhibited high BOD (average values from 12.87-15.96 mg l(-1)), COD (666.67-796.67 mg l(-1)), TDS (500-1466.67mg l(-1)), total hardness (110-835 mg l(-1)), nitrate (1.12-4.97 mg l(-1)), calcium (30.59-271.22 mg l(-1)), phosphate (0.19-0.48 mg l(-1)) and free CO2 (49.13-102.47 mg l(-1)) and low dissolved oxygen (2.63-3.13 mg l(-1)). Heavy metal analysis revealed that the third and fourth wells are free from heavy metal pollution. Coliform test showed bacterial contamination in all the wells. The values of BOD, COD, TDS and phosphate exceeded the maximum permissible limits and the dissolved oxygen was much lower than the desirable limit in all the well waters. Hence all the four well waters are found unsuitable for domestic purposes as it is confirmed by water quality index. The use of waters of open wells in and around the industrial area may cause health hazards to nearby inhabitants.