Tailored Gender-Affirming Hormone Treatment in Nonbinary Transgender Individuals: A Retrospective Study in a Referral Center Cohort.

Purpose: Hormone treatment (HT) is a cornerstone of gender-affirming therapy in transgender and gender nonconforming people. Nonbinary and genderqueer (NBGQ) people, individuals identifying outside the male to female binary, are increasingly recognized. Not all trans people and NBGQ individuals seek full HT. Current guidelines for HT of transgender and gender nonconforming people do not include specific regimens for NBGQ people who seek tailored treatment. We aimed to compare HT prescribed to NBGQ and binary trans people. Methods: We performed a retrospective study in 602 applicants for gender care in 2013-2015 at a referral clinic for gender dysphoria. GenderQueer Identity questionnaires at entry were used to categorize people as NBGQ or binary transgender (BT). Medical records were assessed until the end of 2019 with regard to HT. Results: A total of 113 individuals identified as nonbinary and 489 as BT before the start of HT. NBGQ persons were less likely to receive conventional HT (82% vs. 92%, p=0.004) and more likely to be prescribed tailored HT than BT people (11% vs. 4.7%, p=0.02). None of the NBGQ individuals who received tailored HT had undergone gonadectomy. A subgroup of NBGQ individuals assigned male at birth using exclusively estradiol had similar estradiol and higher testosterone serum concentrations compared with NBGQ individuals using conventional HT. Conclusion: NBGQ individuals more often receive tailored HT compared with BT people. In the future, individualized endocrine counseling may further shape customized HT regimens for NBGQ individuals. For these purposes, qualitative and prospective studies are needed.

The Redesign and Validation of Multimodal Motion-Assisted Memory Desensitization and Reconsolidation Hardware and Software: Mixed Methods, Modified Delphi-Based Validation Study.

BACKGROUND: In recent years, the delivery of evidence-based therapies targeting posttraumatic stress disorder (PTSD) has been the focus of the Departments of Defense in countries such as Canada, the Netherlands, and the United States. More than 66% of military members continue to experience symptoms of PTSD that significantly impact their daily functioning and quality of life after completing evidence-based treatments. Innovative, engaging, and effective treatments for PTSD are needed. Multimodal motion-assisted memory desensitization and reconsolidation (3MDR) is an exposure-based, virtual reality-supported therapy used to treat military members and veterans with treatment-resistant PTSD. Given the demonstrated efficacy of 3MDR in recently published randomized control trials, there is both an interest in and a need to adapt the intervention to other populations affected by trauma and to improve accessibility to the treatment. OBJECTIVE: We aimed to further innovate, develop, and validate new and existing hardware and software components of 3MDR to enhance its mobility, accessibility, feasibility, and applicability to other populations affected by trauma, including public safety personnel (PSP), via international collaboration. METHODS: This study used a modified Delphi expert consultation method and mixed methods quasi-experimental validation with the purpose of software validation among PSP (first responders, health care providers) participants (N=35). A team of international experts from the Netherlands, the United States, and Canada met on the web on a weekly basis since September 2020 to discuss the adoption of 3MDR in real-world contexts, hardware and software development, and software validation. The evolution of 3MDR hardware and software was undertaken followed by a mixed methods software validation study with triangulation of results to inform the further development of 3MDR. RESULTS: This study resulted in the identification, description, and evolution of hardware and software components and the development of new 3MDR software. Within the software validation, PSP participants widely acknowledged that the newly developed 3MDR software would be applicable and feasible for PSP affected by trauma within their professions. The key themes that emerged from the thematic analysis among the PSP included the desire for occupationally tailored environments, individually tailored immersion, and the applicability of 3MDR beyond military populations. CONCLUSIONS: Within the modified Delphi consultation and software validation study, support for 3MDR as an intervention was communicated. PSP participants perceived that 3MDR was relevant for populations affected by trauma beyond military members and veterans. The resulting hardware and software evolution addressed the recommendations and themes that arose from PSP participants. 3MDR is a novel, structured, exposure-based, virtual reality-supported therapy that is currently used to treat military members and veterans with PTSD. Going forward, it is necessary to innovate and adapt 3MDR, as well as other trauma interventions, to increase effectiveness, accessibility, cost-effectiveness, and efficacy among other populations affected by trauma.

Overground and Virtual Reality Gait Speed Are Associated With Atypical Symptom Reporting in Active Duty Service Members With a History of Mild to Moderate Traumatic Brain Injury.

OBJECTIVE: Physical therapy following traumatic brain injury (TBI) can be negatively impacted by psychological symptoms, atypical symptom reporting, and response bias. We examined rates of Symptom Validity Test (SVT) failure in active duty military service members with a history of mild-moderate TBI and its impact on gait speed. SETTING: Intensive Outpatient Program at the National Intrepid Center of Excellence at Walter Reed National Military Medical Center. PARTICIPANTS: Participants were 84 active duty service members with a history of mild-moderate TBI classified as SVT pass ( n = 49) or SVT fail ( n = 35). DESIGN: Retrospective study. MAIN MEASURES: Overground preferred and fast walking speed as well as Computer Assisted Rehabilitation Environment (CAREN) gait speed were recorded. Participants completed the Neurobehavioral Symptom Inventory and the Validity-10 was used to assign patients into the SVT pass and SVT fail groups. Gait speed metrics were compared across these groups and test operating characteristics were calculated. RESULTS: Approximately 42% of the sample was classified into the SVT fail group. All 3 gait speed measures were significantly slower in the SVT fail group than in the SVT pass group ( P s < .001, d s = 0.60-0.80). Gait speed cutoffs for screeners or indicators of atypical reporting were identified. CONCLUSIONS: The potential for response bias is a critical area for the clinician to consider when conducting physical therapy evaluations. Participants in the SVT fail group had slower walking speed on all 3 measures assessed. Several useful cutoffs were identified to serve as screeners or indicators of SVT failure, though these preliminary findings have limitations and need to be replicated.

Examining the Relationship Between Conventional Outcomes and Immersive Balance Task Performance in Service Members With Mild Traumatic Brain Injury.

INTRODUCTION: Mild traumatic brain injury (mTBI) is associated with significant financial cost and reduced military readiness and impacts quality of life for active duty service members (SMs). Post-concussive symptoms can include vestibular impairments, such as chronic dizziness and postural instability, which can be compounded by psychological comorbidities like PTSD. Comprehensive vestibular evaluations are required to assess symptoms and guide clinical decision-making. At the National Intrepid Center of Excellence (NICoE), in addition to traditional vestibular assessments, clinicians can also leverage virtual environments (VEs) in the Computer-Assisted Rehabilitation Environment (CAREN) to further evaluate balance. The objective of this study was to examine the relationship between conventional outcomes and VE performance on immersive balance tasks in the CAREN, determine whether VE performance could predict conventional outcomes, and explore the impact of PTSD. MATERIALS AND METHODS: This retrospective analysis included 112 SMs from the NICoE Intensive Outpatient Program who provided informed consent for their clinical data to be used for research purposes. All had a history of mTBI, underwent vestibular evaluations, and completed immersive balance tasks on the CAREN. Conventional outcomes included the Sensory Organization Test (SOT), Functional Gait Assessment (FGA), Activities Balance Confidence (ABC) Scale, and Dizziness Handicap Inventory (DHI). The PTSD Checklist-Military Version was added to account for behavioral symptoms. Computer-Assisted Rehabilitation Environment outcomes included total time to complete the Balance Cubes VE, with the platform static (BC-Static) and with random platform motion (BC-PM) as well as composite scores for the Shark Hunt VE, with (SH-Recall) and without a recall task (SH-Standard). Statistical analyses included independent t-tests to determine group differences, Pearson's correlations to examine relationships between conventional outcomes and VE performance, and binary logistic regressions to determine if VE performance predicted conventional outcomes based on clinically relevant cutoffs. RESULTS: SMs who took less time to complete BC-Static had better overall balance, indicated by higher ABC, SOT, and FGA scores (P <.001). Those with greater self-reported dizziness, higher DHI scores, took longer to complete BC-Static (P < .05). FGA and SOT, objective gait and balance, were similarly correlated to BC-PM performance (P <.001). SMs with higher SOT scores, better balance, also tended to have higher SH-Standard scores (P <.1). SMs, who were above normative SOT, FGA, and ABC cutoffs, completed BC-Static significantly faster than those below (P <.05). This remained true for BC-PM but was only significant for SOT (P = .004). Performance on BC-Static, BC-PM, and SH-Standard was significant predictors of SOT score. For SMs with comorbid mTBI and PTSD, increased functional disability was observed in conventional outcomes and diminished VE performance was noted. CONCLUSION: Objective balance and gait, SOT and FGA, demonstrated the strongest relationships to immersive VE performance in the CAREN. Our findings suggest that these immersive balance tasks may be effective as an adjunct assessment to examine balance. Future work will focus on moving these VEs from the CAREN to a portable system, which could be more readily utilized in a variety of clinical settings, increasing accessibility.

Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells.

Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1+ cells that infiltrate the neural tissue in a microglia-like manner.

The Effect of Walking on Auditory Localization, Visual Discrimination, and Aurally Aided Visual Search.

OBJECTIVE: The present study was designed to examine the impact that walking has on performance in auditory localization, visual discrimination, and aurally aided visual search tasks. BACKGROUND: Auditory localization and visual search are critical skills that are frequently conducted by moving observers, but most laboratory studies of these tasks have been conducted on stationary listeners who were either seated or standing during stimulus presentation. METHOD: Thirty participants completed three different tasks while either standing still or while walking at a comfortable self-selected pace on a treadmill: (1) an auditory localization task, where they identified the perceived location of a target sound; (2) a visual discrimination task, where they identified a visual target presented at a known location directly in front of the listener; and (3) an aurally aided visual search task, where they identified a visual target that was presented in the presence of multiple visual distracters either in isolation or in conjunction with a spatially colocated auditory cue. RESULTS: Participants who were walking performed auditory localization and aurally aided visual search tasks significantly faster than those who were standing, with no loss in accuracy. CONCLUSION: The improved aurally aided visual search performance found in this experiment may be related to enhanced overall activation caused by walking. It is also possible that the slight head movements required may have provided auditory cues that enhanced localization accuracy. APPLICATION: The results have potential applications in virtual and augmented reality displays where audio cues might be presented to listeners while walking.

The Effects of Blast-Related Neurotrauma on Aurally Aided Visual Search While Standing and Walking.

Service members (SMs) who have suffered mild traumatic brain injury due to blast exposure (b/TBI) often report post-concussive symptoms consistent with auditory, visual, or vestibular impairments even when they score within the normal range on traditional clinical tests of sensory function. One possible explanation for this discrepancy is that patients who score in the low normal range in more than one sensory modality may be severely impaired in tasks that require multisensory integration. This study evaluated unimodal and multimodal sensory performance in SMs with b/TBI and healthy controls by having them conduct four tasks while walking or standing in an immersive virtual environment: an Auditory Localization task (AL) where they moved a cursor to the perceived location of a sound; a Visual Discrimination task (VD) where they distinguished between two visual targets; an Aurally-Aided Visual Search Task (AAVS) where they used an auditory cue to locate and identify a visual target hidden in a field of visual distractors; and a Visual-Only Visual Search task (VOVS) where they located and identified a visual target in a field of distractors with no auditory cue. The results show the b/TBI and healthy control groups performed equivalently in the AL and VD tasks, but that the b/TBI group responded roughly 15% slower in the AAVS task and 50% slower in the VOVS task. Walking had no effect on performance in the visual-only tasks, but both groups responded faster while walking in the AL and AAVS tasks without any reduction in accuracy.

Expanding Clinical Assessment for Traumatic Brain Injury and Comorbid Post-Traumatic Stress Disorder: A Retrospective Analysis of Virtual Environment Tasks in the Computer-Assisted Rehabilitation Environment.

The objective of this study was to determine whether physical performance during virtual environment (VE) tasks in the Computer-Assisted Rehabilitation Environment (CAREN) could differentiate between service members (SMs) with a history of traumatic brain injury (TBI) with and without comorbid post-traumatic stress disorder (PTSD). Data were obtained by independent review of clinical notes, objective outcomes, and validated questionnaires from 214 SMs (208 males) with a history of TBI assessed in the CAREN from 2010 to 2015. Three preliminary VEs acclimatized patients to the CAREN: Balance Balls, weight shifting on a static platform (timed); Balance Cubes, step shifting with and without platform motion (timed); and Continuous Road, flat ambulation (self-selected speed). Multiple regression analyses revealed that patients with comorbid TBI-PTSD were significantly slower in completing the VE tasks than patients without PTSD. Logistic regression showed that the Balance Cubes VE without platform motion significantly predicted diagnostic category (i.e., no PTSD vs. comorbid PTSD). In conclusion, in SMs with a history of TBI, physical performance on the CAREN effectively distinguished those with comorbid PTSD, as their performance was significantly slower than SMs without PTSD. These results portray the potential of the CAREN as a novel assessment tool in SMs with a history of TBI.

Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.

The nuclear transactive response DNA-binding protein 43 (TDP-43) undergoes relocalization to the cytoplasm with formation of cytoplasmic deposits in neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Pathogenic mutations in the TDP-43-encoding TARDBP gene in familial ALS as well as non-mutant human TDP-43 have been utilized to model FTD/ALS in cell culture and animals, including mice. Here, we report novel A315T mutant TDP-43 transgenic mice, iTDP-43(A315T), with controlled neuronal over-expression. Constitutive expression of human TDP-43(A315T) resulted in pronounced early-onset and progressive neurodegeneration, which was associated with compromised motor performance, spatial memory and disinhibition. Muscle atrophy resulted in reduced grip strength. Cortical degeneration presented with pronounced astrocyte activation. Using differential protein extraction from iTDP-43(A315T) brains, we found cytoplasmic localization, fragmentation, phosphorylation and ubiquitination and insolubility of TDP-43. Surprisingly, suppression of human TDP-43(A315T) expression in mice with overt neurodegeneration for only 1 week was sufficient to significantly improve motor and behavioral deficits, and reduce astrogliosis. Our data suggest that functional deficits in iTDP-43(A315T) mice are at least in part a direct and transient effect of the presence of TDP-43(A315T). Furthermore, it illustrates the compensatory capacity of compromised neurons once transgenic TDP-43 is removed, with implications for future treatments.

If You Build It, They Will Come, But What Will Wounded Warriors Experience? Presence in the CAREN.

Military service members (SMs) are surviving complex battlefield injuries at higher rates than ever before. Cutting-edge technologies are increasingly being employed to improve assessment and treatment of these complex injuries. The Computer Assisted Rehabilitation Environment (CAREN) is a comprehensive immersive environment, featuring a treadmill, curved panoramic screen, audio array, and infrared cameras to capture movement. While the CAREN has been progressively incorporated in treatment and research, little has been reported regarding participants' subjective experiences, particularly in relation to the signature wounds of the Iraq and Afghanistan wars, traumatic brain injury (TBI) and posttraumatic stress disorder (PTSD). Here we report participant presence, in a cohort of SMs with complex, frequently dual-diagnosis injuries (N=148; 95% TBI; 58% PTSD) engaging in CAREN-driven treatment. Using a presence questionnaire, participants rated aspects of the CAREN on a 7-point Likert scale and a presence score was calculated. The average presence score was 46.83 (SD=6.04; possible score range 7-63), with 95% of participants reporting scores >36. Those with motion sickness and eye discomfort reported lower presence, whereas those with PTSD reported higher presence. Presence did not vary according to TBI severity. Overall, SMs with complex injuries experience presence in the CAREN. However, presence may be adversely affected by CAREN-associated symptoms such as motion sickness.

Academy of Nutrition and Dietetics and National Kidney Foundation: revised 2014 Standards of Practice and Standards of Professional Performance for registered dietitian nutritionists (competent, proficient, and expert) in nephrology nutrition.

Compelling evidence indicates that the incidence of chronic kidney disease (CKD) is increasing because of an aging population and a higher prevalence of cardiovascular disease, diabetes, and hypertension. Nutrition management of patients with CKD requires early disease recognition, appropriate interpretation of the markers and stages of CKD, and collaboration with other health care practitioners. Better management of CKD can slow its progression, prevent metabolic complications, and reduce cardiovascular related outcomes. Caring for patients with CKD necessitates specialized knowledge and skills to meet the challenges associated with this growing epidemic. The Academy of Nutrition and Dietetics Renal Dietitians Practice Group and the National Kidney Foundation Council on Renal Nutrition, with guidance from the Academy of Nutrition and Dietetics Quality Management Committee, have updated the 2009 Standards of Practice in Nutrition Care and Standards of Professional Performance as a tool for registered dietitian nutritionists working in nephrology nutrition to assess their current skill levels and to identify areas for additional professional development in this practice area. The Standards of Practice apply to the care of patients/clients with kidney disease. The Standards of Professional Performance consist of six domains of professionalism, including: Quality in Practice, Competence and Accountability, Provision of Services, Application of Research, Communication and Application of Knowledge, and Utilization and Management of Resources. Within each standard, specific indicators provide measurable action statements that illustrate how nephrology nutrition principles can be applied to practice. The indicators describe three skill levels (ie, competent, proficient, and expert) for registered dietitian nutritionists working in nephrology nutrition.

Academy of Nutrition and Dietetics and National Kidney Foundation: revised 2014 standards of practice and standards of professional performance for registered dietitian nutritionists (competent, proficient, and expert) in nephrology nutrition.

Compelling evidence indicates that the incidence of chronic kidney disease (CKD) is increasing because of an aging population and a higher prevalence of cardiovascular disease, diabetes, and hypertension. Nutrition management of patients with CKD requires early disease recognition, appropriate interpretation of the markers and stages of CKD, and collaboration with other health care practitioners. Better management of CKD can slow its progression, prevent metabolic complications, and reduce cardiovascular related outcomes. Caring for patients with CKD necessitates specialized knowledge and skills to meet the challenges associated with this growing epidemic. The Academy of Nutrition and Dietetics Renal Dietitians Practice Group and the National Kidney Foundation Council on Renal Nutrition, with guidance from the Academy of Nutrition and Dietetics Quality Management Committee, have updated the 2009 Standards of Practice in Nutrition Care and Standards of Professional Performance as a tool for registered dietitian nutritionists working in nephrology nutrition to assess their current skill levels and to identify areas for additional professional development in this practice area. The Standards of Practice apply to the care of patients/clients with kidney disease. The Standards of Professional Performance consist of six domains of professionalism, including: Quality in Practice, Competence and Accountability, Provision of Services, Application of Research, Communication and Application of Knowledge, and Utilization and Management of Resources. Within each standard, specific indicators provide measurable action statements that illustrate how nephrology nutrition principles can be applied to practice. The indicators describe three skill levels (ie, competent, proficient, and expert) for registered dietitian nutritionists working in nephrology nutrition.

Transplante renal e gestação / Kidney transplantation and pregnancy

O transplante renal é considerado o melhor tratamento para a doença renal terminal. Essa terapia tem avançado em diversos aspectos, contribuindo para que pacientes transplantadas em idade fértil possam ter uma gestação bem-sucedida. Foi conduzida revisão da literatura nas bases de dados Periódico Capes e PubMed/MEDLINE no período entre 1998 e 2013, visando melhor compreensão e acompanhamento de gestantes submetidas a transplante renal.O sucesso dessas gestações depende de vários fatores, incluindo planejamento pré-concepção minucioso baseado na saúde geral da paciente, função do enxerto, história de rejeição, pressão arterial sistêmica, proteinúria e ultrassom do enxerto. Há relatos de nascidos vivos em torno de 70% dessas gestações, com mortalidade perinatal de 10%, deterioração do enxerto em 5% dos casos, e média temporal entre o transplante e a gestação de aproximadamente três anos. As principais complicações foram maiores taxas de parto cesáreo, hipertensão arterial sistêmica e parto pré-termo. Embora a frequência de complicações perinatais descrita nessa população seja elevada, o transplante renal não é considerado uma contraindicação para a gestação. É necessário melhor avaliação dos efeitos dos agentes imunossupressores sobre o feto e o recém-nato para garantir segurança do uso dessas drogas durante a gestação e a amamentação.(AU)

Renal transplantation is considered the best treatment for end-stage renal disease. This therapy has advanced in many ways, contributing to transplanted patients of childbearing age can have a successful pregnancy. It was conducted a literature review in Capes Journal and PubMed/MEDLINE database between the period of 1998 and 2013, aiming at better understanding and monitoring of pregnant women who underwent renal transplantation. The success of these gestations depends on diverse factors, including a thorough preconception planning based on the patient general health, graft function, history of rejection, systemic blood pressure, proteinuria and graft ultrasound. There are reports of live births in about 70% of the gestations, with a perinatal mortality of 10%, graft deterioration in 5% of the cases, and temporal average between the transplantation and the gestation of approximately three years. The main complications were higher rates of cesarean delivery, systemic arterial hypertension and preterm birth. Although the frequency of perinatal complications described in this population is high, renal transplantation is not considered a contraindication to pregnancy. It is necessary better assessment of the imunossupressors agents effects on the fetus and newborns to ensure safety of the use of these drugs during pregnancy and breastfeeding.(AU)

Single nucleotide variants (SNVs) define senescence-accelerated SAMP8 mice, a model of a geriatric condition.

One of the major challenges in neurodegenerative research is modeling systemic aging. Here, senescence-accelerated mice such as the multigenic SAMP8 (senescence accelerated prone 8) mice are useful as they are characterized by an early manifestation of senescence that includes a shortened lifespan and impaired brain and immune functions. While SAMP8 mice are widely used tools to address aging and neurodegenerative conditions such as Alzheimer's disease (AD), the underlying gene mutations are not known. To make the SAMP8 strain a more versatile and useful research tool, we performed exome sequencing, using SAMR1 (senescence accelerated mouse resistant 1) mice as controls. We identified 51 SNVs (single nucleotide variants) that discriminate SAMP8 from SAMR1 mice. Using the prediction tool Polyphen2, we were able to subdivide the SNVs into four categories: splice variants, probably damaging, possibly damaging, and benign. Of these genes, a significant fraction is predicted to be expressed in the brain. Our data present these genes for a more detailed analysis in aging and neurodegeneration studies. They underscore the usefulness of SAMP8 mice as an animal model to study fundamental mechanisms of both aging and the pathogenesis of AD.

Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.

BACKGROUND: Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. RESULTS: We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. CONCLUSIONS: Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling.

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2beta, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.

Synthesis of C8-N-acetylarylamine 2'-dG-adducts and their site-specifically incorporation into oligonucleotides.

Beside the predominately found C8-NH-arylamine-dG damages also C8-N-acetylarylamine-lesions of 2'-dG seems to play an important role in the induction of the chemical carcinogenesis. A new synthetic pathway leading to these DNA-adducts using different aromatic amines have been developed. The C8-dG-adducts were converted into the corresponding phosphoramidites and incorporated site-specifically into oligonucleotides giving damaged DNA-strands. With damaged DNA hybrids studies with respect to their thermal stability (UV melting temperature analysis) and circular dichroism were performed.

Characterisation of inosine monophosphate dehydrogenase expression during retinal development: differences between variants and isoforms.

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigations of the recently described retinal type I variants. Inosine monophosphate dehydrogenase was present in all immature cells throughout the retina during embryonic and neonatal development. Following eye opening and cell differentiation its distribution was restricted to the photoreceptors and bipolar cells, becoming prominent in Müller cells with aging. Type II was present in early, developing retinae whilst type I was undetectable. An isoform switch occurred around P10, after which the type I variants, type Ialpha and type Igamma, were the major forms. Functional investigations indicate type Igamma has greater catalytic activity compared with other variants and isoforms. Finally, all forms of type I show an increased propensity to form intracellular macrostructures compared to type II and these structures appear to be regulated in response to changing intracellular GTP levels. Collectively these data demonstrate that (i) type I does not play a role in early retinal development, (ii) type Igamma has greater activity and (iii) there are differences between type I and type II isoforms. These observations are consistent with the aetiology of retinitis pigmentosa and raise the possibility that programmed expression of specific inosine monophosphate dehydrogenase proteins may have arisen to meet the requirements of the cellular environment.

Seminal fluid characterization for male fertility and prostate cancer: kallikrein-related serine proteases and whole proteome approaches.

The kallikrein-related ( KLK) protease gene family encodes a subgroup of 15 serine proteases that includes prostate-specific antigen (PSA) or KLK3, the well-known biomarker for prostate cancer. PSA is also a major component of seminal fluid. To date, 10 other KLK serine proteases have been documented as present in seminal fluid (KLKs 1, 2, 4, 5, 6, 8, 10, 11, 13, and 14) and, like PSA, have the potential to contribute to male fertility, either directly or indirectly, by means of their proteolytic activity on seminal coagulum proteins. These KLK enzymes arise predominantly from the glandular epithelium of the prostate and are secreted into the lumen of the prostatic ducts that empty into the urethra upon ejaculation. Given their prostatic origin, they are also being considered increasingly as diagnostic/prognostic targets for prostate cancer. This article reviews the literature on seminal fluid PSA and more recent reports on the detection of other KLKs enzymes in this milieu, and their potential roles in male fertility and prostate cancer. We also discuss recent efforts to determine the proteomic profile of seminal fluid to identify new biomarkers for prostate disease.