Field-Based Planetary Protection Operations for Melt Probes: Validation of Clean Access into the Blood Falls, Antarctica, Englacial Ecosystem.

Subglacial environments on Earth offer important analogs to Ocean World targets in our solar system. These unique microbial ecosystems remain understudied due to the challenges of access through thick glacial ice (tens to hundreds of meters). Additionally, sub-ice collections must be conducted in a clean manner to ensure sample integrity for downstream microbiological and geochemical analyses. We describe the field-based cleaning of a melt probe that was used to collect brine samples from within a glacier conduit at Blood Falls, Antarctica, for geomicrobiological studies. We used a thermoelectric melting probe called the IceMole that was designed to be minimally invasive in that the logistical requirements in support of drilling operations were small and the probe could be cleaned, even in a remote field setting, so as to minimize potential contamination. In our study, the exterior bioburden on the IceMole was reduced to levels measured in most clean rooms, and below that of the ice surrounding our sampling target. Potential microbial contaminants were identified during the cleaning process; however, very few were detected in the final englacial sample collected with the IceMole and were present in extremely low abundances (∼0.063% of 16S rRNA gene amplicon sequences). This cleaning protocol can help minimize contamination when working in remote field locations, support microbiological sampling of terrestrial subglacial environments using melting probes, and help inform planetary protection challenges for Ocean World analog mission concepts.

Spliced HLA-bound peptides: a Black Swan event in immunology.

Peptides that bind to and are presented on the cell surface by human leucocyte antigen (HLA) molecules play a critical role in adaptive immunity. For a long time it was believed that all the HLA-bound peptides were generated through simple proteolysis of linear sequences of cellular proteins, and therefore are templated in the genome and proteome. However, evidence for untemplated peptide ligands of HLA molecules has accumulated during the last two decades, with a recent global analysis of HLA-bound peptides suggesting that a considerable proportion of HLA-bound peptides are potentially generated through splicing/fusion of discontinuous peptide segments from one or two distinct proteins. In this review, we will evaluate recent discoveries and debates on the contribution of spliced peptides to the HLA class I immunopeptidome, consider biochemical rules for splicing and the potential role of these spliced peptides in immune recognition.

Transmission of Xylella fastidiosa to Grapevine by the Meadow Spittlebug.

There is little information available on Xylella fastidiosa transmission by spittlebugs (Hemiptera, Cercopoidea). This group of insect vectors may be of epidemiological relevance in certain diseases, so it is important to better understand the basic parameters of X. fastidiosa transmission by spittlebugs. We used grapevines as a host plant and the aphrophorid Philaenus spumarius as a vector to estimate the effect of plant access time on X. fastidiosa transmission to plants; in addition, bacterial population estimates in the heads of vectors were determined and correlated with plant infection status. Results show that transmission efficiency of X. fastidiosa by P. spumarius increased with plant access time, similarly to insect vectors in another family (Hemiptera, Cicadellidae). Furthermore, a positive correlation between pathogen populations in P. spumarius and transmission to plants was observed. Bacterial populations in insects were one to two orders of magnitude lower than those observed in leafhopper vectors, and population size peaked within 3 days of plant access period. These results suggest that P. spumarius has either a limited number of sites in the foregut that may be colonized, or that fluid dynamics in the mouthparts of these insects is different from that in leafhoppers. Altogether our results indicate that X. fastidiosa transmission by spittlebugs is similar to that by leafhoppers. In addition, the relationship between cell numbers in vectors and plant infection may have under-appreciated consequences to pathogen spread.

Subglacial Lake Whillans microbial biogeochemistry: a synthesis of current knowledge.

Liquid water occurs below glaciers and ice sheets globally, enabling the existence of an array of aquatic microbial ecosystems. In Antarctica, large subglacial lakes are present beneath hundreds to thousands of metres of ice, and scientific interest in exploring these environments has escalated over the past decade. After years of planning, the first team of scientists and engineers cleanly accessed and retrieved pristine samples from a West Antarctic subglacial lake ecosystem in January 2013. This paper reviews the findings to date on Subglacial Lake Whillans and presents new supporting data on the carbon and energy metabolism of resident microbes. The analysis of water and sediments from the lake revealed a diverse microbial community composed of bacteria and archaea that are close relatives of species known to use reduced N, S or Fe and CH4 as energy sources. The water chemistry of Subglacial Lake Whillans was dominated by weathering products from silicate minerals with a minor influence from seawater. Contributions to water chemistry from microbial sulfide oxidation and carbonation reactions were supported by genomic data. Collectively, these results provide unequivocal evidence that subglacial environments in this region of West Antarctica host active microbial ecosystems that participate in subglacial biogeochemical cycling.

A comprehensive analysis of peptides presented by HLA-A1.

Human leukocyte antigen (HLA)-A1 is one of the most common Caucasian HLA-A alleles. Here, we describe the comprehensive analysis of the HLA-A*01:01 ligand repertoire with the identification of 4735 naturally processed and presented peptides derived from 2477 source proteins. We found HLA-A*01:01 bound an equivalent number of ligands of 9 or 10 amino acids in length as well as being remarkably tolerant of even longer peptides. Indeed close to half of the HLA-A1 bound peptides identified ranged between 11 and 13 amino acids in length. These longer peptides contained the strong canonical motif of and acidic E/D residue at position 3 (P3) and Y at the C-terminus (CΩ), a motif that was still apparent in peptides of up to 18 amino acids in length. The identification of this large database of natural ligands will facilitate the refinement of predictive algorithms particularly with respect to longer peptide ligands.

Serum phosphorylated neurofilament-heavy chain levels in multiple sclerosis patients.

OBJECTIVES: We evaluated whether the measurement of serum phosphorylated neurofilament heavy chain (pNF-H) titre is likely to be a valid biomarker of axonal injury in multiple sclerosis (MS). METHODS: Serum pNF-H concentrations were measured by ELISA in cases with relapsing-remitting (RR)-MS (n=81), secondary progressive (SP) MS (n=13) and primary progressive (PP)-MS; n=6) MS; first demyelinating event (FDE; n=82); and unaffected controls (n=135). A subset of MS cases (n=45) were re-sampled on one or multiple occasions. The Multiple Sclerosis Severity Score (MSSS) and MRI measures were used to evaluate associations between serum pNF-H status, disease severity and cerebral lesion load and activity. RESULTS: We confirmed the presence of pNF-H peptides in serum by ELISA. We showed that a high serum pNF-H titre was detectable in 9% of RR-MS and FDE cases, and 38.5% of SP-MS cases. Patients with a high serum pNF-H titre had higher average MSSS scores and T2 lesion volumes than patients with a low serum pNF-H titre. Repeated sampling of a subset of MS cases showed that pNF-H levels can fluctuate over time, likely reflecting temporal dynamics of axonal injury in MS. CONCLUSIONS: A subset of FDE/MS cases was found to have a high serum pNF-H titre, and this was associated with changes in clinical outcome measures. We propose that routine measurement of serum pNF-H should be further investigated for monitoring axonal injury in MS.

A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides.

The human B lymphoblastoid cell line C1R is widely regarded as human leukocyte antigen-A (HLA-A)/HLA-B negative and is therefore frequently exploited as a recipient cell line to study HLA class I functions. However, the normal levels of HLA-C*04:01 often hamper the investigation of introduced HLA class I allomorphs, which is particularly evident in sensitive applications such as mass spectrometry. Here we describe the comprehensive analysis of endogenous HLA-C*04:01 ligands expressed on the surface of C1R cells to (i) define a large sequence dataset of HLA-C*04:01 ligands, to (ii) refine the HLA-C*04:01 peptide-binding motif and (iii) to provide a resource that allows discrimination between peptides bound to introduced HLA class I subtypes and to the endogenous HLA-C*04:01 molecules.

Methadone use in pregnancy: evidence of progression in the severity of addiction.

The number of opiate users is well documented; however, the severity of opiate use has received little attention. This retrospective study in a North of England hospital updates the progression in the severity of addiction in pregnancy. Patients treated were reviewed and the doses of prescribed methadone documented. Historical data were also used for comparison. The severity in addiction in pregnancy was assessed by the woman's drug usage expressed as the daily dose of prescribed methadone at the end of pregnancy. From 2001 to 2008 there was an increase in the mean dose of methadone prescribed at delivery from 28.2 ml/day in 2001 to 57.9 ml/day in 2008. Historically, the use was 27.3 ml/day in 1992-1996 and 32.4 ml/day in 1997-2003. No trend was noted in the number of pregnant users. In conclusion, we observed no recent increase in the number of methadone users presenting, but the severity of drug usage in pregnancy has increased.

Eutectic mixture of local anaesthetics (EMLA) 5% cream as a primary dressing on a painful lower leg ulcer.

Patients living with chronic leg ulceration may frequently experience moderate to severe wound-related pain, which may not be alleviated by oral analgesics alone. Poorly controlled leg ulcer pain can prevent timely and effective wound management strategies being implemented, and increase wound healing times. Furthermore, patients with poorly controlled leg ulcer pain can experience continuous pain, which significantly affects quality of life. This case report introduces an innovative way of using the eutectic mixture of local anaesthetics (EMLA) 5% cream to reduce wound-related pain, reduce oral analgesic intake, and improve health-related quality of life for a patient with a painful, chronic lower leg ulcer.

Expressive language skills in Chinese Singaporean preschoolers with nonsyndromic cleft lip and/or palate.

OBJECTIVE: The main objective of the present study was to examine THE EXPRESSIVE LANGUAGE SKILLS and obtain a prevalence estimate of expressive language IMPAIRMENT (not skills) in Chinese Singaporean preschoolers with nonsyndromic cleft lip and/or palate (CLP). METHODS: A group of 43 Chinese Singaporean preschoolers aged 3 to 6 years with a diagnosis of nonsyndromic CLP was assessed using the Singapore English Action Picture Test (SEAPT). The SEAPT is an English Language screening tool standardised on typically developing English-Mandarin Chinese Singaporean preschoolers that assesses expressive vocabulary and grammatical usage. A grammar and/or information score below the 20(th) percentile on the SEAPT is indicative of an expressive language impairment. In addition, the medical records of this cohort were examined retrospectively for documentation of surgical timings, audiological history, articulation and resonance. RESULTS: Based on the results of the SEAPT, 33% of the preschoolers with CLP were identified as having a-possible expressive language impairment. Hence, the likelihood that a child with CLP with normal cognitive functioning will have an expressive language impairment is between 3.9 to 12.7 times more likely than in the general population. There was no statistical significance when comparisons were made between dominant language groups or CLP groups on SEAPT measures of information and grammar content. Significantly more males than females were identified with language difficulties, relative to the sex ratio in the sample. No significance was found for the other participant variables. CONCLUSIONS: The findings of this study suggest that Chinese Singaporean preschoolers with CLP have more difficulty in the expressive use of grammar and vocabulary than their peers of typical development, with significantly more males affected than females. As language performance was not related to hearing, articulation or resonance; these early results suggest that a comprehensive investigation of cognition, literacy and family aggregation of communication disorders is urgently warranted to study other possible aetiologies for language impairment in children with CLP in Singapore.

The influence of maternal opiate use in pregnancy on second trimester biochemical markers for Down syndrome.

OBJECTIVE: The object of this study is to examine the influence of maternal opiate use on the levels of second trimester biochemical markers for Down syndrome. Maternal opiate use is known to be associated with problems of placental origin and it is possible that the secretion of alpha-feto protein (AFP), free-beta human chorionic gonadotrophin (HCG) and unconjugated oestriol (UE) differs from that of a normal population. METHOD: Seventy nine women who used opiates in pregnancy were compared to a control group of seventy nine women who did not use opiates and their adjusted marker levels analysed. RESULTS: The adjusted median MoM in the opiate and control groups respectively were: AFP (1.00 vs 0.94), HCG (0.95 vs 1.04) and UE (0.96 vs 1.02), with no significant difference between these groups. CONCLUSION: This study suggests that the current practice of calculating the risk of Down syndrome from second trimester biochemistry in women using opiate can be performed using data derived from a normal population.

Intraoperative ketamine may influence persistent pain following knee arthroplasty under combined general and spinal anaesthesia: a pilot study.

We report the findings of a randomised controlled triple-blind pilot study of intraoperative ketamine infusion combined with spinal anaesthesia on the prevalence of persisting post surgical pain following total knee arthroplasty surgery. Twelve patients were randomised to receive either ketamine or placebo in association with spinal anaesthesia for total knee arthroplasty. All patients also received general anaesthesia. More patients were pain-free at six months in the ketamine group (three of five) compared to the control group (two of seven). Perioperative data collected during the study suggested that the addition of intraoperative ketamine might also improve the quality of recovery. Although no statistical analysis was undertaken due to the small numbers, these preliminary findings suggest that the use of intraoperative systemic ketamine in association with spinal anaesthesia for the reduction of persisting post surgical pain deserves further study.

The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.

Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.

Immunodominance hierarchies and gender bias in direct T(CD8)-cell alloreactivity.

Allogeneic solid organ transplantation often occurs across multiple donor-recipient HLA mismatches with consequent risk of allograft rejection. However, there is growing evidence that not all HLA mismatches are equivalent in their stimulation of allogeneic T cells making it important to determine which of these might be more significant as predictors of allograft rejection. To this end, we used defined antigen-presenting cell (APC) transfectants expressing single MHC-I allotypes as target cells that could discriminate the relative contribution of individual mismatched MHC-I allotypes to direct T-cell alloreactivity. We demonstrate remarkably reproducible patterns of immunodominance in reactivity across mismatched MHC-I allotypes. These patterns are HLA context-dependent, partly reflecting alloantigenic competition in responder cell responses. In strong alloresponses, we also observed an increased percentage of alloreactive T(CD8) cells in female responders, regardless of the stimulator gender, highlighting HLA-independent factors in the potency of the alloresponse. This approach provides a potential measure of specific alloreactive T cells that could be used in clinical practice for selection of donors, assessment of posttransplant outcomes, modulation of immunosuppression and detection of rejection episodes.

The molecular basis of cross-reactivity in the Australian Snake Venom Detection Kit (SVDK).

The Snake Venom Detection Kit (SVDK) is of major medical importance in Australia, yet it has never been rigorously characterised in terms of its sensitivity and specificity, especially when it comes to reports of false-negative and false-positive results. This study investigates reactions and cross-reactions of five venoms the SVDK is directed against and a number of purified toxins. Snakes showing the closest evolutionary relationships demonstrated the lowest level of cross-reactivity between groups. This was, instead, far more evident between snakes that are extraordinarily evolutionary separated. These snakes: Pseudechis australis, Acanthophis antarcticus and Notechis scutatus, in fact displayed more false-positive results. Examination of individual toxin groups showed that phospholipase A(2)s (PLA(2)s) tends to react strongly and display considerable cross-reactivity across groups while the three-finger toxins (3FTx) reacted poorly in all but the Acanthophis well. The hook effect was evident for all venoms, particularly Oxyuranus scutellatus. The results of this study show considerable variation in toxin detection, with implications in further development of venom detection, both in Australia and other countries.

The Fate of Xylella fastidiosa in Vineyard Weeds and Other Alternate Hosts in California.

The fate of Xylella fastidiosa, the bacterium that causes Pierce's disease of grape, was assessed in 29 species of plants associated with vineyards in California's San Joaquin Valley. Bacterial populations and movement in greenhouse-grown plants were measured 1, 3, and 9 weeks after mechanical and insect inoculation. X. fastidiosa was recovered in 27 of 29 species in greenhouse tests, with common sunflower (Helianthus annuus), cocklebur (Xanthium strumarium), annual bur-sage (Ambrosia acanthicarpa), morning glory (Ipomoea purpurea), horseweed (Conyza canadensis), sacred datura (Datura wrightii), poison hemlock (Conium maculatum), and fava bean (Vicia faba cv. Aquadulce) being infected in more than 50% of inoculation attempts. Twenty-three species supported bacterial populations in excess of 104 CFU/g of plant tissue. X. fastidiosa populations increased for 9 weeks after inoculation in six species, and were static or declined in 16 species. Although the blue-green sharpshooter (Graphocephala atropunctata) was more efficient than mechanical inoculation for infecting plants, median populations and systemic movement for the two methods did not differ significantly. X. fastidiosa colonization of greenhouse and field-grown plants was compared in five alternate hosts. X. fastidiosa was recovered from 23 and 34% of field-grown plants, compared with 49 and 67% of greenhouse-grown plants, in the winter and summer, respectively, with at least 10 times fewer bacteria in field plants in the winter. Although X. fastidiosa has a wide host plant range, its fate in most species is variable and heavily influenced by field conditions.

Vector Transmission of Xylella fastidiosa to Dormant Grape.

Homalodisca coagulata (Say) is a sharpshooter leafhopper vector of the bacterial plant pathogen Xylella fastidiosa. Introduced into California about 15 years ago, this insect triggered recent outbreaks of Pierce's disease of grapevine in the state. H. coagulata has been observed feeding on dormant grapevines during the winter, raising the possibility of X. fastidiosa transmission during that season. We tested whether H. coagulata can acquire X. fastidiosa from and inoculate the bacterium to dormant grape (Vitis vinifera) in the laboratory and in the field. Usually, >90% of H. coagulata survived on dormant plants in the laboratory and field. Field experiments showed that H. coagulata can inoculate X. fastidiosa into dormant plants, yet field acquisition experiments did not result in transmission. Transmission to dormant plants during the winter is a potential problem in California vineyards adjacent to citrus groves or other habitats with large overwintering populations of H. coagulata. Because dormant plants have positive root pressure, our findings provide evidence that X. fastidiosa transmission does not require negative pressure in plant xylem to be inoculated into plants.

Immunoproteomics: Mass spectrometry-based methods to study the targets of the immune response.

The mammalian immune system has evolved to display fragments of protein antigens derived from microbial pathogens to immune effector cells. These fragments are typically peptides liberated from the intact antigens through distinct proteolytic mechanisms that are subsequently transported to the cell surface bound to chaperone-like receptors known as major histocompatibility complex (MHC) molecules. These complexes are then scrutinized by effector T cells that express clonally distributed T cell receptors with specificity for specific MHC-peptide complexes. In normal uninfected cells, this process of antigen processing and presentation occurs continuously, with the resultant array of self-antigen-derived peptides displayed on the surface of these cells. Changes in this peptide landscape of cells act to alert immune effector cells to changes in the intracellular environment that may be associated with infection, malignant transformation, or other abnormal cellular processes, resulting in a cascade of events that result in their elimination. Because peptides play such a crucial role in informing the immune system of infection with viral or microbial pathogens and the transformation of cells in malignancy, the tools of proteomics, in particular mass spectrometry, are ideally suited to study these immune responses at a molecular level. Here we review recent advances in the studies of immune responses that have utilized mass spectrometry and associated technologies, with specific examples from collaboration between our laboratories.

Novel strategy for identification of candidate cytotoxic T-cell epitopes from human preproinsulin.

We describe a strategy for identifying ligands of human leukocyte antigen (HLA) class I molecules based on a peptide library-mediated in vitro assembly of recombinant class I molecules. We established a microscale class I assembly assay and used a capture ELISA to quantify the assembled HLA-peptide complexes. The identity of the bound ligands was then deduced by mass spectrometry. In this method, HLA complexes assembled in vitro in the presence of components of a mixture of peptides were immunoprecipitated and the bound peptide(s) identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This process of epitope extraction is robust and can be used with complex mixtures containing in excess of 300 candidate ligands. A library of overlapping peptides representing all potential octamers, nonamers and decamers from human preproinsulin was synthesized using unique library chemistry. Peptides from the library were used to initiate assembly of recombinant HLA-B8, HLA-B15 and HLA-A2, facilitating the identification of candidate T-cell epitopes from preproinsulin.