The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis.

Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using ß2-microglobulin (ß2m) as a model system, focusing on D76N-ß2m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting ß2m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite ß-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-ß2m) we show that residue 76 has a unique ability to drive ß2m aggregation in vivo and in vitro. Using a randomly mutated D76N-ß2m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-ß2m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.

Mechanisms behind the varying severity of Aleutian mink disease virus: Comparison of three farms with a different disease status.

Aleutian mink disease virus (AMDV) is distributed widely among mink farms and wild mustelids despite ongoing attempts to stop the spread. The severity of Aleutian disease (AD) varies from subclinical to fatal but the reasons for its varying severity are complex and unclear. Recently, breeding of tolerant mink has drawn attention as the possible solution to reduce the effects of AD in farms. The aim of this study was to gather information on the effects of breeding based on overall health, production traits, and antibody titer on AD severity by comparing a positive farm (farm 1) that has been breeding for tolerance in mink to an infected farm without tolerance selection, and an AMDV-free farm. During the 2.5-year follow-up, the mink in farm 1 remained mostly free of clinical AD, had normal pelt quality and litter size, and had low virus copy numbers in tissues and low antibody titers in ELISA. In histopathological studies, most of the farm 1 mink had no/mild lesions in their kidneys. 29-43% of the mink were ELISA negative but PCR positive throughout the follow-up and frequent changes in virus strains and coinfections were observed. Several differences in gene expression between animals from different farms were also detected. These results indicate that the disease burden of AMDV can be reduced, with seemingly normal health and production rates, despite continual circulation of ADMV in cases where eradication attempts are unsuccessful.

Corrigendum: The Impact of a Dissonance-Based Eating Disorders Intervention on Implicit Attitudes to Thinness in Women of Diverse Sexual Orientations.

[This corrects the article DOI: 10.3389/fpsyg.2019.02611.].

The Impact of a Dissonance-Based Eating Disorders Intervention on Implicit Attitudes to Thinness in Women of Diverse Sexual Orientations.

Dissonance-based body image programs have shown long-term effectiveness in preventing eating disorders and reducing risk factors for eating disorders in women. Here we report on the potential for one such intervention to impact on implicit attitudes toward thinness as well as an explicit measure of eating attitudes, across a sexually diverse group of young women. The Succeed Body Image Programme was adapted to remove heteronormative assumptions and was delivered to a final sample of 56 undergraduate women who reported their sexual orientation as either "predominantly heterosexual" (our term; 1 or 2 on a 7-point Kinsey scale, n = 38) or non-heterosexual (3-7 on the Kinsey scale, n = 18). Before and after the intervention, they completed the Eating Attitudes Test-26, and an associative reaction time task based on the Implicit Association Test, in which bodies of low and higher weight were paired with socially desirable or undesirable traits. A total of 37 predominantly heterosexual women completed a control intervention in which they read NHS leaflets on eating disorders and healthy weight. Results showed that the intervention made predominantly heterosexual participants less prone, versus control, to associating thinness with positive traits on the IAT and all women completing the intervention reported a lower level of disordered eating attitudes at post- than pre-test. Non-heterosexual women, however, showed a non-significant increase in thin-bias on the IAT, perhaps due to their low baseline. These results imply that intensive dissonance-based programs can change attitudes at the automatic, implicit level as well as merely giving women tools to overcome those implicit attitudes.

Restriction Fragment Length Polymorphism-based Genotyping of Toxoplasma gondii from Autopsy-Proven Cases of Acquired Immunodeficiency Syndrome-associated Cerebral Toxoplasmosis.

CONTEXT: Published data on genetic characterization of Toxoplasma gondii (T.gondii) from clinical cases of toxoplasmosis from India is lacking. AIMS: The present study was aimed at identifying genetic types of T. gondii in fatal cases of cerebral toxoplasmosis (CT) associated with HIV, from India. SETTINGS AND DESIGN: Archived tissues of CT were obtained postmortem from 25 acquired immunodeficiency syndrome patients between 2000 and 2014. SUBJECTS AND METHODS: Direct amplification of eight different loci, namely, SAG1, 5'-3'SAG2, Alt. SAG2, SAG3, BTUB, GRA6, C22-8, and L358 followed by restriction fragment length polymorphism was used to genotype the parasite. RESULTS: The canonical Types I, II, or III were not found in our study. More than 96% of the cases harbored atypical genotypes-likely recombinants of the canonical types; one case closely corresponded to Type II genotype. CONCLUSIONS: Thus, a majority of T. gondii causing CT in South India belonged to a noncanonical lineage. These nonarchetypal genotypes differed from the conventional Types I, II, and III and caused devastating severity in patients with CT in the background of HIV. These results are a step further to deciphering the population genetics of this important zoonotic parasitic infection in Indian patients, information that has thus far been lacking.

Genetic characterization of Toxoplasma gondii from autopsy proven cases of AIDS associated cerebral toxoplasmosis in South India.

Toxoplasma gondii (T.gondii) infection can be devastating in the immunodeficient causing high morbidity and mortality. Due to limited availability of both diagnostic facilities and Highly Active Antiretroviral Therapy (HAART), toxoplasmosis continues to be a significant problem amongst Acquired Immuno Deficiency Syndrome (AIDS) patients in India. While scanty literature is available on T. gondii isolates in animals in India, little is known about the genetic diversity of the parasite in humans. Therefore, the present study investigated the genetic diversity of T. gondii in 25 confirmed cases of cerebral toxoplasmosis developing on the background of human immunodeficiency virus (HIV) infection/AIDS. PCR DNA sequencing was performed at four important genetic loci of T. gondii: BTUB, GRA6, alternative SAG2 (alt SAG2) and SAG3 on DNA from tissues obtained at postmortem. The amplified products from all the cases were successfully sequenced except at one locus for one case. Results of the present study suggest that majority of the patients (22/25; 88%) in South India are infected with strains that are recombinants of type II/III and/or strains representing T. gondii different from the archetypal lineages I, II, and III. In addition, clonal types III, MAS, and MAS variant genotypes were encountered. No clonal type I or II was seen in the present study. In addition, variants were observed at alt SAG2 and SAG3 but BTUB and GRA6 were highly conserved. Single nucleotide polymorphisms were observed mainly at two loci which are coding for surface antigens at alt SAG2 and SAG3. In conclusion, the present study reveals genetic diversity in India amongst strains of T. gondii from clinical cases of toxoplasmosis which is in accordance with other recent studies showing a high rate of genetic diversity in this parasite across the globe. There is a need to genotype T. gondii from different forms of toxoplasmosis in humans in India.

Allelic forms of the knob associated histidine-rich protein gene of Plasmodium falciparum.

The knob associated histidine-rich protein (KAHRP) gene was cloned and sequenced from two Indian isolates of Plasmodium falciparum, Pf3-92 and Pf29-92. These isolates showed major sequence differences in the C-terminal repeat domain of KAHRP. However, the biologically important domains such as spectrin-actin binding region remained highly conserved. The PCR amplification of a variable C-terminal repeat domain from the clinical isolates of P. falciparum, from Rajasthan epidemic, showed the presence of multiple alleles of KAHRP gene. The presence of multiple alleles indicates the existence of several P. falciparum strains in India. This should be taken into account for future malaria control strategies such as molecular therapy and vaccines.

Cytotoxic effects of 125I-labeled PBZr ligand PK 11195 in prostatic tumor cells: therapeutic implications.

The effect of [125I]PK 11195 was examined in human prostatic tumor cells (DU 145) in culture and compared with Na[125I] and non-radioactive PK 11195. [125I]PK 11195 was clearly cytocidal. The data for dose-related cell survival with [125I]PK 11195 showed a linear relationship. Na[125I] or non-labeled PK 11195 at similar concentrations did not lead to any cell killing. The uptake of [125I]PK 11195 and [3H]PK 11195 in cells was very similar. Fragmentation of DNA measured by agarose gel electrophoresis showed that exposure of DU 145 cells to [1251]PK 11195 for 1, 4 or 24 h caused no fragmentation. These results indicate that nuclear DNA is not the prime binding site for [125I]PK 11195, which is consistent with the presence of specific peripheral benzodiazepine receptors (PBZr) in the mitochondria. The cell killing effect of [125I]PK 11195 suggests the use of PBZr ligand for radiotherapy.

Outcome of laminectomy for civilian gunshot injuries of the terminal spinal cord and cauda equina: review of 88 cases.

Case records of 88 patients with low-velocity gunshot injuries of the terminal spinal cord and cauda equina treated by laminectomy at Cook County Hospital between 1969 and 1987 were reviewed. Sixty-one patients were operated upon within 72 hours of injury, 29 of whom (47.5%) experienced neurological improvement or pain relief. Twenty-seven patients were operated upon at a later time for associated injuries, 13 of whom (48.1%) experienced neurological improvement or pain relief. When laminectomy was delayed for more than 2 weeks, either arachnoid adhesions (15%) or occult abscesses (17%) were observed. From this review as well as from the literature, it appears that the timing of laminectomy for gunshot injuries of the thoracolumbar and lumbosacral spine is not essential to neurological recovery. It appears, however, that adequate debridement of these injuries, performed as soon as the patient is stable from any associated injuries, may help to mitigate the late sequelae of arachnoiditis, infection, and pain syndromes in the lower extremities.

Anterior cervical debridement and strut-grafting for osteomyelitis of the cervical spine.

A retrospective review of the surgical experience in treating 18 patients with osteomyelitis of the cervical spine is reported. The patients ranged in age from 20 to 60 years and all complained of neck pain upon admission. Ten patients had a prior history of intravenous drug abuse, three had previously suffered penetrating injuries of the neck, and one had an extraspinal site of osteomyelitis. Bacteria were isolated in 13 cases and tuberculosis in three. Neurological abnormalities were present in over one-half of the patients, consisting of myelopathy (nine cases) or radiculopathy (four cases). Plain cervical spine films and polytomography demonstrated vertebral and end-plate destruction, spinal instability, and increased paravertebral soft-tissue shadow in all cases. Computerized tomography and, more recently, magnetic resonance imaging have proven helpful in detecting bone involvement and the presence of epidural extension associated with cervical osteomyelitis. The risk of vertebral body collapse, kyphosis, and myelopathy in the osteomyelitic cervical spine has standardized the management of this problem in this institution to consist of skeletal traction, needle aspiration or blood culture for organism identification, anterior cervical debridement, autogenous iliac graft fusion, and intravenous administration of antibiotics. Spinal stability and neurological improvement were achieved in all 18 patients.

A comparative study of serological and biochemical methods for strain differentiation of foot-and-mouth disease type A viruses.

Three serological and three biochemical methods were used to compare five field isolates of foot-and-mouth disease virus (FMDV) from Western India with nine reference vaccine strains and five field isolates from other countries. The serological tests (liquid-phase ELISA and virus neutralization) were able to distinguish between the three reference vaccine strains examined, but the five Indian field isolates reacted poorly with antisera produced against these vaccine strains. Analysis of monoclonal antibody (mAb) data was difficult to interpret although clearly the field isolate A/IND/5/87 reacted to a lesser extent with one of the mAb panels (A10/Holland/42) than the other four Indian isolates. The A22/Iraq/24/64 mAbs did not react with any of the Indian field isolates and only significantly with one of the reference vaccine strains, A/IND/57/79. Polyacrylamide gel electrophoresis distinguished the reference vaccine strains from each other and from the field isolates. Additionally, one of the Indian isolates (A/IND/5/87) could be differentiated from the other four. Electrofocusing showed similarities between the reference vaccine strain A22/Iraq/24/64 and three of the Indian field isolates (A/IND/1/87, A/IND/2/87 and A/IND/3/87), however, A/IND/4/87 and A/IND/5/87 were distinct. Nucleotide sequencing showed that the isolates A/IND/1/87, A/IND/2/87 and A/IND/3/87 were very closely related to each other and related to A/IND/4/87, however, A/IND/5/87 was different.

Primary chondrosarcoma of the breast: a case report.

Mammary sarcomas are uncommon tumors. Of these pure chondrosarcoma without any other area of epithelial or mesenchymal differentiation feature as even rarer. This excludes tumors like malignant cystosarcoma phyllodes and metaplastic carcinoma where malignant cartilaginous areas may be present. Further primary chondrosarcoma arises from breast stroma and not from underlying bone or cartilage. Only five cases of pure and primary chondrosarcoma have been reported so far. The sixth case is reported here.

Seasonal prevalence and succession of rice field breeding mosquitoes of central Gujarat.

Studies on seasonal prevalence and succession of mosquitoes in rice fields, revealed the dominance of Anopheles culicifacies and An. subpictus in newly transplanted fields during early months of rice cultivation, which was later replaced by the species like An. annularis, An. barbirostris, An. nigerrimus and An. tessellatus with the growth in plant height. Among culicines, except Cx. quinquefasciatus other species were prevalent during growing and later stages of the rice crop. Larval density was found inversely proportional to the height of plants, whereas species diversity maximized during growing phase due to equitable number of specimen among species.

The Chennai Declaration: a roadmap to tackle the challenge of antimicrobial resistance.

"A Roadmap to Tackle the Challenge of Antimicrobial Resistance - A Joint meeting of Medical Societies in India" was organized as a pre-conference symposium of the 2 nd annual conference of the Clinical Infectious Disease Society (CIDSCON 2012) at Chennai on 24 th August. This was the first ever meeting of medical societies in India on issue of tackling resistance, with a plan to formulate a road map to tackle the global challenge of antimicrobial resistance from the Indian perspective. We had representatives from most medical societies in India, eminent policy makers from both central and state governments, representatives of World Health Organization, National Accreditation Board of Hospitals, Medical Council of India, Drug Controller General of India, and Indian Council of Medical Research along with well-known dignitaries in the Indian medical field. The meeting was attended by a large gathering of health care professionals. The meeting consisted of plenary and interactive discussion sessions designed to seek experience and views from a large range of health care professionals and included six international experts who shared action plans in their respective regions. The intention was to gain a broad consensus and range of opinions to guide formation of the road map. The ethos of the meeting was very much not to look back but rather to look forward and make joint efforts to tackle the menace of antibiotic resistance. The Chennai Declaration will be submitted to all stake holders.