Current and future practices in surgical retraction.

Retraction of tissues and anatomical structures is an essential component of all forms of surgery. The means by which operative access is gained through retraction are many and diverse. In this article, the various forms of retraction methods currently available are reviewed, with special reference to hand held, self-retaining and compliant techniques. The special challenges posed by laparoscopic surgery are considered and future developments in new retraction techniques are anticipated.

Incidence of cerebrovascular accident following head and neck free tissue transfer surgery.

The aim of this study was to determine the incidence of postoperative cerebrovascular accident (CVA) following head and neck free tissue transfer and to identify predictive risk factors. A retrospective audit was performed of patients who underwent head and neck reconstructive surgery at Queen Elizabeth University Hospital between 2009 and 2020. The patient records were analysed to identify those who developed CVA within 30 days after surgery. A total of 1109 patients underwent head and neck free tissue transfer surgery, including 1048 neck dissection procedures. Of these, 78.6% had one or more identified risk factors for perioperative stroke. Five patients (0.45%) developed postoperative CVA. The results showed that CVA correlated to patients with hypercholesterolemia (P = 0.007). This study demonstrates the safety of free tissue transfer. Despite underlying co-morbidities and risk factors, the incidence of CVA is low following surgery and manipulation of the major vasculature of the neck.

Spinal motoneurons respond aberrantly to serotonin in a rabbit model of cerebral palsy.

Cerebral palsy (CP) is caused by a variety of factors that damage the developing central nervous system. Impaired motor control, including muscle stiffness and spasticity, is the hallmark of spastic CP. Rabbits that experience hypoxic-ischemic (HI) injury in utero (at 70-80% gestation) are born with muscle stiffness, hyperreflexia, and, as recently discovered, increased serotonin (5-HT) in the spinal cord. To determine whether serotonergic modulation of spinal motoneurons (MNs) contributes to motor deficits, we performed ex vivo whole cell patch clamp in neonatal rabbit spinal cord slices at postnatal day (P) 0-5. HI MNs responded to application of α-methyl 5-HT (a 5-HT 1 /5-HT 2 receptor agonist) and citalopram (a selective 5-HT reuptake inhibitor) with hyperpolarization of persistent inward currents and threshold voltage for action potentials, reduced maximum firing rate, and an altered pattern of spike frequency adaptation while control MNs did not exhibit any of these responses. To further explore the differential sensitivity of MNs to 5-HT, we performed immunohistochemistry for inhibitory 5-HT 1A receptors in lumbar spinal MNs at P5. Fewer HI MNs expressed the 5-HT 1A receptor compared to age-matched controls. This suggests many HI MNs lack a normal mechanism of central fatigue mediated by 5-HT 1A receptors. Other 5-HT receptors (including 5-HT 2 ) are likely responsible for the robust increase in HI MN excitability. In summary, by directly exciting MNs, the increased concentration of spinal 5-HT in HI rabbits can cause MN hyperexcitability, muscle stiffness, and spasticity characteristic of CP. Therapeutic strategies that target serotonergic neuromodulation may be beneficial to individuals with CP. Key points: After prenatal hypoxia-ischemia (HI), neonatal rabbits that show hypertonia are known to have higher levels of spinal serotoninWe tested responsivity of spinal motoneurons (MNs) in neonatal control and HI rabbits to serotonin using whole cell patch clampMNs from HI rabbits showed a more robust excitatory response to serotonin than control MNs, including hyperpolarization of the persistent inward current and threshold for action potentials, larger post-inhibitory rebound, and less spike frequency adaptation Based on immunohistochemistry of lumbar MNs, fewer HI MNs express inhibitory 5HT 1A receptors than control MNs, which could account for the more robust excitatory response of HI MNs. These results suggest that after HI injury, the increased serotonin could trigger a cascade of events leading to muscle stiffness and altered motor unit development.

Quality and quantity of data recovered from massively parallel sequencing: Examples in Asparagales and Poaceae.

PREMISE OF THE STUDY: Genome survey sequences (GSS) from massively parallel sequencing have potential to provide large, cost-effective data sets for phylogenetic inference, replace single gene or spacer regions as DNA barcodes, and provide a plethora of data for other comparative molecular evolution studies. Here we report on the application of this method to estimating the molecular phylogeny of core Asparagales, investigating plastid gene losses, assembling complete plastid genomes, and determining the type and quality of assembled genomic data attainable from Illumina 80-120-bp reads. METHODS: We sequenced total genomic DNA from samples in two lineages of monocotyledonous plants, Poaceae and Asparagales, on the Illumina platform in a multiplex arrangement. We compared reference-based assemblies to de novo contigs, evaluated consistency of assemblies resulting from use of various references sequences, and assessed our methods to obtain sequence assemblies in nonmodel taxa. KEY RESULTS: Our method returned reliable, robust organellar and nrDNA sequences in a variety of plant lineages. High quality assemblies are not dependent on genome size, amount of plastid present in the total genomic DNA template, or relatedness of available reference sequences for assembly. Phylogenetic results revealed familial and subfamilial relationships within Asparagales with high bootstrap support, although placement of the monotypic genus Aphyllanthes was placed with moderate confidence. CONCLUSIONS: The well-supported molecular phylogeny provides evidence for delineation of subfamilies within core Asparagales. With advances in technology and bioinformatics tools, the use of massively parallel sequencing will continue to become easier and more affordable for phylogenomic and molecular evolutionary biology investigations.

Biodiversity assessment: state-of-the-art techniques in phylogenomics and species identification.

PREMISE OF THE STUDY: Biodiversity assessment is the first step in protecting the complete range of morphological and genetic diversity of species on Earth, and in reaching the goals of conservation biology. Assessment begins with identifying organisms that make up biological communities and understanding evolutionary histories. Scientific advancements in molecular sequencing can help clarify and provide support for identifications. Massively parallel DNA sequencing technologies are being used to sequence complete genomes of model organisms; however, this resource has not been fully used for species identifications. Animal researchers commonly use one mitochondrial region, and groups of plant scientists have proposed numerous combinations of two or three chloroplast markers as genomic identifiers. Yet, nearly as many studies have reported that the proposed regions are uninformative in some plant groups and at various taxonomic levels. METHODS: We propose a combination of whole (or nearly whole) chloroplast genomes, mitochondrial genes, and nuclear repeat regions for both species identifications and phylogenetic analyses, obtained from a simple total DNA extraction and one run on massively parallel DNA sequencing machines. KEY RESULTS: We have recovered both coding and noncoding sequences from multiple genetic sources, providing genomic information for comparisons within and between multiple taxonomic levels. CONCLUSIONS: In combination with morphological and other data, this abundance of genomic information will have a broad range of applications, including not only helping conservation biologists understand ecosystem biodiversity, but also understanding the evolutionary histories of organisms, mending damaged landscapes, and investigating interactions of plants with pollinators and pests.

Survival after the initiation of combination therapy in patients with pulmonary arterial hypertension: an Australian collaborative report.

BACKGROUND: Several cellular pathways are implicated in the pathogenesis of pulmonary arterial hypertension (PAH) and attempts to arrest disease progression with a single drug would not be expected to succeed in the medium term. In clinical practice, combination therapy is often used in patients deteriorating on monotherapy, despite the absence of firm evidence from randomized controlled controls. METHODS: From January 2005 to August 2009, 112 patients with World Health Organisation Functional Class (FC) II-IV PAH deteriorating on monotherapy received non-parenteral combination therapy at six Australian PAH expert hospitals. Combination therapy included bosentan, sitaxentan, ambrisentan, iloprost and sildenafil. Data were prospectively collected for survival status, 6-min walk distance, FC and echocardiographic parameters at the start of monotherapy through to commencement of combination therapy and at 6-monthly intervals thereafter. RESULTS: After varying periods of monotherapy (18.7±13.4onths), survival estimates on combination therapy were 88%, 71% and 61% for the additional 1, 2 and 3years respectively. Survival on dual therapy in patients with idiopathic PAH/familial PAH was 93% at 1year and 79% at 2years, and for scleroderma-related PAH, 72% at 1 year and 48% at year 2 after initiation of combination therapy. In survivors, dual therapy reversed the deterioration in FC, from 3.1±0.6 on monotherapy to 2.2±0.6 at 12months. Similarly, dual therapy improved 6-min walk distance from 316±119m to 406±129m at 12months, and sequential echocardiography demonstrated a fall in pulmonary artery systolic pressure and improved right ventricular function. CONCLUSIONS: Dual non-parenteral therapy appears safe and effective and should be considered for PAH patients who are deteriorating on monotherapy to improve long-term outcomes.

Operative tactics in floor of mouth and tongue cancer resection - the importance of imaging and planning.

Complete tumour resection (R0 margin) is an axiom of surgical oncology. Oral cancer ablation is challenging, due to anatomical, functional, and aesthetic considerations. R0 margin is strongly linked to better survival outcomes with great variation in the R0 % across units. This is commonly attributed to disease biology. Without disputing the importance of biological characteristics, we contend that image-based anatomical surgical planning has an important role to play in achieving complete resection. Here, we present our approach utilising cross-sectional imaging, anatomical characteristics and spatial awareness in planning resections for floor of mouth (FOM) and oral tongue cancers. We highlight the challenge of controlling the deep tumour margin lingual to mandible due to anterior vector constraints and emphasise the importance of resecting the genial muscles in a planned fashion and that any rim resection should be obliquely sagittal. In resecting lateral FOM tumours, assessing extension to the parapharyngeal fat is crucial; and mandibular rim resection at a sagittal plane below the mylohyoid line is often required. Assessing the proximity of the contralateral neurovascular pedicle, pre-epiglottic space and hyoid bone are crucial parameters to determine the extent of tongue tumour resection. Our cohort included 173 patients with FOM SCC and 299 patients with tongue SCC. Six patients (3.5%) from the FOM group and eight patients (3%) from the tongue group had involved (R1) margins following surgery. This was associated with local relapse (p<0.05). In conclusion, we demonstrate that image-based planning can aid achieving R0 resections and reduce disease relapse.

Applying the British Association of Oral and Maxillofacial Surgeons quality outcomes metrics to a UK oncology and reconstructive surgery service - benchmarking the data.

The British Association of Oral and Maxillofacial Surgery is soon to implement the Quality Outcomes in Oral in Maxillofacial Surgery (QOMS) to provide a platform for quality management across the specialty in the UK. The initial oncology and reconstruction audits for QOMS involves data collection on specific procedures and metrics. The aim of this report is to determine their appropriateness using extant audit datasets in our institution that overlap substantially with the QOMS audits. Pre-existing datasets comprising information on patients treated for oral cavity SCC with curative intent were analysed. Data on surgical margins, lymphadenectomy lymph node yield, delay between surgery and adjuvant radiotherapy, duration of hospital stay, and complications including flap failures were analysed. All statistical analyses were performed with SPSS 25. Run charts describing longitudinal data were generated using SPC for Excel version 6. Twenty three patients (3.1%) of 701 resections had a positive surgical margin reported. Seventeen (4.3%) of patients had less than 18 LNs in the ND specimen analysed. Mean time to start date of adjuvant therapy was 62 days. Only 9% of patients commenced adjuvant therapy within 6 weeks. The median duration of stay was 18 days. In 1153 free flaps a failure rate of 4.3% was identified. A total of 1349 complications (CD I-V) were recorded in the 1111 patients undergoing major surgery with free flap reconstruction. The QOMS selected metrics for oncology and reconstruction are clinically relevant, readily measurable, and likely to be actionable by the surgical team.

Distinctive termini characterize two families of human endogenous retroviral sequences.

Human DNA contains many copies of endogenous retroviral sequences. Characterization of molecular clones of these structures reveals the existence of two related families. One family consists of full-length (8.8 kilobases) proviral structures, with typical long terminal repeates (LTR's). The other family consists of structures, which contain only 4.1 kilobases of gag-pol sequences, bounded by a tandem array of imperfect repeats 72 to 76 base pairs in length. Typical LTR sequences that exist as solitary elements in the genome were cloned and characterized.

Phylogenetic utility of 141 low-copy nuclear regions in taxa at different taxonomic levels in two distantly related families of rosids.

Angiosperm systematics has progressed to the point where it is now expected that multiple, independent markers be used in phylogenetic studies. Universal primers for amplifying informative regions of the chloroplast genome are readily available, but in the faster-evolving nuclear genome it is challenging to discover priming sites that are conserved across distantly related taxa. With goals including the identification of informative markers in rosids, and perhaps other angiosperms, we screened 141 nuclear primer combinations for phylogenetic utility in two distinct groups of rosids at different taxonomic levels-Psiguria (Cucurbitaceae) and Geraniaceae. We discovered three phylogenetically informative regions in Psiguria and two in Geraniaceae, but none that were useful in both groups. Extending beyond rosids, we combined our findings with those of another recent effort testing these primer pairs in Asteraceae, Brassicaceae, and Orchidaceae. From this comparison, we identified 32 primer combinations that amplified regions in representative species of at least two of the five distantly related angiosperm families, giving some prior indication about phylogenetic usefulness of these markers in other flowering plants. This reduced set of primer pairs for amplifying low-copy nuclear markers along with a recommended experimental strategy provide a framework for identifying phylogenetically informative regions in angiosperms.

Diagnosis of preclinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies.

In most sheep infected with a transmissible spongiform encephalopathy (tse) the disease-associated prion protein (PrP(d)) accumulates in tissues of the lymphoreticular system, suggesting that it might be detected in biopsy specimens. A procedure has been developed to obtain biopsy specimens of rectal mucosa in which PrP(d) has been detected by immunohistochemistry in preclinically infected sheep of all susceptible PrP genotypes. It is probable that PrP(d) increases with the age of sheep or period of incubation. PrP(d) was detectable approximately halfway through the incubation period, with sheep of some PrP genotypes showing positive results earlier than others. For a preclinical diagnosis, the risk of a false negative result was approximately 9 per cent for samples containing 10 follicles, a figure that was reached in 87 per cent of the biopsies. The rectal biopsies had the same sensitivity and time of onset of PrP(d) accumulation as biopsies of the palatine tonsil, but provided larger numbers of follicles. The procedure is simple and quick, does not require dedicated specific instruments, sedation or general anaesthesia, and can be performed repeatedly on the same sheep without detrimental effects to either the animal or the number of follicles obtained.

Solid-state framing camera operating in interferometric mode.

A high speed solid-state framing camera has been developed which can operate in interferometric mode. This camera measures the change in the index of refraction of a semiconductor when x-rays are incident upon it. This instrument uses an x-ray transmission grating/mask in front of the semiconductor to induce a corresponding phase grating in the semiconductor which can then be measured by an infrared probe beam. The probe beam scatters off of this grating, enabling a measure of the x-ray signal incident on the semiconductor. In this particular instrument, the zero-order reflected probe beam is attenuated and interfered with the diffracted orders to produce an interferometric image on a charge coupled device camera of the phase change induced inside the semiconductor by the incident x-rays.

Adoptive transfer of lymphocytes sensitized to the major surface glycoprotein of Pneumocystis carinii confers protection in the rat.

Pneumocystis carinii is a major opportunistic pathogen and a leading cause of morbidity in patients with AIDS. CD4+ cells have been shown to be important in host defenses against P. carinii, but the antigen(s) involved with this response have not been identified. We undertook the present study to determine whether the major surface glycoprotein (MSG) of P. carinii contains epitopes that can elicit a protective cellular immune response. Spleen cells and purified CD4+ cells isolated from Lewis rats, pulsed 1-4 d with MSG, and injected into corticosteroid-treated Lewis rats with pneumocystosis resulted in significant reduction in the P. carinii burden, as judged by organism quantitation and lung histology. The protective response demonstrated by the donor cells was dependent on previous exposure to P. carinii, cell concentration, and time of incubation with MSG. In addition, reconstitution with MSG-specific CD4+ cells resulted in an early hyperinflammatory response within the lungs of these animals with a high percentage of mortality. Thus, in this model, MSG can elicit an immune response mediated by CD4+ cells, which has a harmful as well as helpful effect on the host, and these responses occur despite the presence of corticosteroids.

Discrimination of sheep susceptible and resistant to transmissible spongiform encephalopathies by an haplotype specific monoclonal antibody.

In the present report, the selective detection of sheep PrP haplotypes by monoclonal antibody 2A11 is described. It is showed that the substitution of glutamine by arginine but not by histidine at ovine PrP position 171 abolishes completely the recognition of either PrP(c) or PrP(d) by mAb 2A11, in such a way that the application of this antibody allows the unambiguous discrimination of R(171) homozygotes. On the basis of the high resistance to classical scrapie and bovine spongiform encephalophaty (BSE) infection associated to the R(171) PrP haplotype, animals bearing the ARR allele are currently selected within the scrapie national plan initiated in Great Britain. A 2A11-based immuno enzymatic test have been developed and evaluated using a panel of plasma and sera from sheep of different PrP genotypes and breeds. The test allows the efficient discrimination of R(171) homozygotes, R(171) heterozygotes and non-R(171) carriers, therefore offering a rapid, cheap and easy to use alternative method to select sheep for their resistance to scrapie.

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Do negative or positive emotions differentially impact mortality after adult stem cell transplant?

Multiple diverse biomedical variables have been shown to affect outcome after hematopoietic stem cell transplantation (HSCT). Whether psychosocial variables should be added to the list is controversial. Some empirical reports have fueled skepticism about the relationship between behavioral variables and HSCT survival. Most of these reports have methodological shortcomings. Their samples were small in size and included heterogeneous patient populations with different malignant disease and disease stages. Most data analyses did not control adequately for biomedical factors using multivariate analyses. The pre-transplant evaluations differed from study to study, making cross-study generalizations difficult. Nevertheless, a few recently published studies challenge this skepticism, and provide evidence for deleterious effects of depressive symptomatology on HSCT outcome. This mini review integrates the new data with previously reviewed data, focusing on the differential impact of negative and positive emotional profiles on survival. Pre-transplant negative emotional profiles are associated with worse survival in the long term, whereas pre-transplant optimism about transplant appears to affect survival in the short term. These data have practical implications for transplant teams. Pre-transplant psychological evaluation should assess for specific adverse behavioral risk factors, particularly higher levels of depression and lower levels of optimistic expectations about transplant. Transplant centers should develop collaborative studies to further test the effects of these adverse behavioral risk factors, and run multicenter hypothesis-driven clinical trials of psychological intervention protocols. Such studies should aim to better define pragmatics of assessment and intervention (timing, assessment tools, personnel), and evaluate their contribution to improving outcome after transplant.

Inactivation of transmissible spongiform encephalopathy agents during the manufacture of dicalcium phosphate from bone.

Dicalcium phosphate was prepared from industrial crushed bone artificially contaminated with transmissible spongiform encephalopathy agents in two experiments carried out in an accurately scaled-down laboratory model of the industrial manufacturing process. In one experiment, 10 g of mouse brain infected with the 301V strain of mouse-passaged bovine spongiform encephalopathy agent was added to the crushed bone; in the other experiment, 10 g of hamster brain infected with the 263K strain of hamster-passaged scrapie agent was added. Samples of the infectious brain and dried dicalcium phosphate were assayed for the amount of 301V or 263K infectivity present. The titre of infectivity of the 301V-infected brain was 10(7.7) intracerebral ID50/g; that of the 263K-infected brain was 10(8.0) intracerebral ID50/g. The titres of the dried samples of dicalcium phosphate were 10(2.5) ID50/g in the experiment spiked with 301V and 10(2.7) ID50/g in the experiment spiked with 263K. The calculated clearance factors were 10(3.9) for the experiment with 301V and 10(3.8) for the experiment with 263K.