Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma

Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 105 and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies

Pretreatment malnutrition and quality of life - association with prolonged length of hospital stay among patients with gynecological cancer: a cohort study

Background Length of hospital stay (LOS) is a surrogate marker for patients' well-being during hospital treatment and is associated with health care costs. Identifying pretreatment factors associated with LOS in surgical patients may enable early intervention in order to reduce postoperative LOS. Methods This cohort study enrolled 157 patients with suspected or proven gynecological cancer at a tertiary cancer centre (2004-2006). Before commencing treatment, the scored Patient Generated - Subjective Global Assessment (PG-SGA) measuring nutritional status and the Functional Assessment of Cancer Therapy-General (FACT-G) scale measuring quality of life (QOL) were completed. Clinical and demographic patient characteristics were prospectively obtained. Patients were grouped into those with prolonged LOS if their hospital stay was greater than the median LOS and those with average or below average LOS. Results Patients' mean age was 58 years (SD 14 years). Preoperatively, 81 (52%) patients presented with suspected benign disease/pelvic mass, 23 (15%) with suspected advanced ovarian cancer, 36 (23%) patients with suspected endometrial and 17 (11%) with cervical cancer, respectively. In univariate models prolonged LOS was associated with low serum albumin or hemoglobin, malnutrition (PG-SGA score and PG-SGA group B or C), low pretreatment FACT-G score, and suspected diagnosis of cancer. In multivariable models, PG-SGA group B or C, FACT-G score and suspected diagnosis of advanced ovarian cancer independently predicted LOS. Conclusions Malnutrition, low quality of life scores and being diagnosed with advanced ovarian cancer are the major determinants of prolonged LOS amongst gynecological cancer patients. Interventions addressing malnutrition and poor QOL may decrease LOS in gynecological cancer patients

The NOURISH randomised control trial: Positive feeding practices and food preferences in early childhood - a primary prevention program for childhood obesity

Background Primary prevention of childhood overweight is an international priority. In Australia 20-25% of 2-8 year olds are already overweight. These children are at substantially increased the risk of becoming overweight adults, with attendant increased risk of morbidity and mortality. Early feeding practices determine infant exposure to food (type, amount, frequency) and include responses (eg coercion) to infant feeding behaviour (eg. food refusal). There is correlational evidence linking parenting style and early feeding practices to child eating behaviour and weight status. A focus on early feeding is consistent with the national focus on early childhood as the foundation for life-long health and well being. The NOURISH trial aims to implement and evaluate a community-based intervention to promote early feeding practices that will foster healthy food preferences and intake and preserve the innate capacity to self-regulate food intake in young children. Methods/Design This randomised controlled trial (RCT) aims to recruit 820 first-time mothers and their healthy term infants. A consecutive sample of eligible mothers will be approached postnatally at major maternity hospitals in Brisbane and Adelaide. Initial consent will be for re-contact for full enrolment when the infants are 4-7 months old. Individual mother- infant dyads will be randomised to usual care or the intervention. The intervention will provide anticipatory guidance via two modules of six fortnightly parent education and peer support group sessions, each followed by six months of regular maintenance contact. The modules will commence when the infants are aged 4-7 and 13-16 months to coincide with establishment of solid feeding, and autonomy and independence, respectively. Outcome measures will be assessed at baseline, with follow up at nine and 18 months. These will include infant intake (type and amount of foods), food preferences, feeding behaviour and growth and self-reported maternal feeding practices and parenting practices and efficacy. Covariates will include sociodemographics, infant feeding mode and temperament, maternal weight status and weight concern and child care exposure. Discussion Despite the strong rationale to focus on parents’ early feeding practices as a key determinant of child food preferences, intake and self-regulatory capacity, prospective longitudinal and intervention studies are rare. This trial will be amongst to provide Level II evidence regarding the impact of an intervention (commencing prior to age 12 months) on children’s eating patterns and behaviours. Trial Registration: ACTRN1260800005639

Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs

Comorbid substance abuse and brain morphology in recent-onset psychosis

The aim of the presented study was to compare schizophrenia and schizoaffective patients early in the course of the disease with and without comorbid substance abuse disorder (SUD vs. NSUD) with regard to brain morphology. In a prospective design 41 patients (20 SUD vs. 21 NSUD) diagnosed as recent-onset schizophrenia or schizoaffective disorder consecutively admitted to hospital received standardized psychopathological evaluation (BPRS, SANS, MADRS, CGI, GAF) and MRI scanning with volumetric measurement of superior temporal gyrus (STG), amygdala-hippocampal complex, and cingulum. Patients with SUD (primarily cannabis) were significantly younger, predominantly male and had a lower socioeconomic status. Despite less attentional impairment (SANS subscore) and elevated anxiety/depression (BPRS subscore) in patients with SUD compared to NSUD, no other psychopathological differences could be detected. There were no differences in the assessed temporolimbic brain morphology between the two subgroups. In conclusion, in this study substance abuse in recent-onset psychosis had no effect on brain morphology and the earlier onset of psychosis in patients with comorbid SUD could not be explained by supposed accentuated brain abnormalities in temporolimbic regions