Deletion of CD44 promotes adipogenesis by regulating PPARγ and cell cycle-related pathways.

CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

Canine Pyothorax: Comparison of Culture and Susceptibility Results to the BSAVA PROTECT ME Poster and Other Published Recommended Antimicrobial Use Guidelines.

The most common bacterial isolates in dogs with pyothorax include mixed anaerobes, Enterobacteriaceae (especially Escherichia coli), Pasteurella spp., Streptococcus spp., and Staphylococcus spp. A fluoroquinolone with amoxicillin (±clavulanate) or a fluoroquinolone with clindamycin are the most commonly recommended empirical antimicrobials whilst pending bacterial culture of the pleural effusion. The aim of this study is to review and compare the pleural effusion culture and antimicrobial susceptibility results to the PROTECT ME poster and other published antimicrobial use guidelines. The medical records of 53 dogs diagnosed with pyothorax between 2014 and 2020 at two veterinary referral centres were reviewed. Information, including culture and susceptibility results, was assessed. Antimicrobial susceptibility panels varied; susceptibility to a particular antibiotic was calculated as a percentage of isolates tested against the same antibiotic. A total of 30 of 53 dogs (57.7%) had a positive pleural fluid culture. The most common isolates were Pasteurella species (23.3%), Escherichia coli (23.3%), and mixed anaerobes (20%). From the aerobic isolates, 73-83% were susceptible to a fluoroquinolone, 14/19 (74%) to amoxicillin, and 20/22 (91%) to potentiated amoxicillin. Resistance to clindamycin was documented in 9/13 (69%) aerobic isolates, with all Gram-negative bacteria (9/9) being resistant. The combination of potentiated amoxicillin with marbofloxacin would have been appropriate in most of the dogs (75-92.9%). This study shows a high rate of resistance to clindamycin, which is not a suitable option for monotherapy and may be less effective in combination therapy compared to potentiated amoxicillin.

Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach.

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

Engineering Geobacter pili to produce metal:organic filaments.

The organized self-assembly of conductive biological structures holds promise for creating new bioelectronic devices. In particular, Geobacter sulfurreducens type IVa pili have proven to be a versatile material for fabricating protein nanowire-based devices. To scale the production of conductive pili, we designed a strain of Shewanella oneidensis that heterologously expressed abundant, conductive Geobacter pili when grown aerobically in liquid culture. S. oneidensis expressing a cysteine-modified pilin, designed to enhance the capability to bind to gold, generated conductive pili that self-assembled into biohybrid filaments in the presence of gold nanoparticles. Elemental composition analysis confirmed the filament-metal interactions within the structures, which were several orders of magnitude larger than previously described metal:organic filaments. The results demonstrate that the S. oneidensis chassis significantly advances the possibilities for facile conductive protein nanowire design and fabrication.

Splenectomy in the management of primary immune-mediated hemolytic anemia and primary immune-mediated thrombocytopenia in dogs.

BACKGROUND: Current reports about the use of splenectomy for the management of immune-mediated hemolytic anemia (IMHA) or immune-mediated thrombocytopenia (ITP) or both in dogs are limited. OBJECTIVES: To retrospectively describe the use of splenectomy as part of the management for IMHA, ITP, and concurrent IMHA and severe thrombocytopenia (CIST) in dogs. It was hypothesized that splenectomy would be beneficial in allowing for reduction of dose of immunosuppressive drugs or discontinuation in 1 or more of these groups. ANIMALS: Seventeen client-owned dogs (7 with IMHA, 7 with ITP, and 3 with CIST) were identified across 7 UK-based referral hospitals from a study period of 2005 to 2016. METHODS: Data were collected retrospectively via questionnaires and included information about diagnosis, management and treatment response before and after splenectomy. Based on clinical outcome, treatment with splenectomy as part of the management protocol was classified as either successful or unsuccessful. RESULTS: Six of 7 dogs with ITP were managed successfully with splenectomy as part of their management protocol (3 complete and 3 partial responses), although 1 subsequently developed suspected IMHA. Of the 7 dogs with IMHA, splenectomy was part of a successful management protocol in 4 dogs (2 complete and 2 partial responses). In the CIST group, 1 case (1/3) responded completely to management with splenectomy as part of the management protocol. CONCLUSIONS AND CLINICAL IMPORTANCE: Splenectomy was considered successful and well tolerated in most cases of isolated ITP. Whether there is a benefit of splenectomy in cases of IMHA and CIST could not be determined in the current study.

Radiation-induced neuroinflammation: a potential protective role for poly(ADP-ribose) polymerase inhibitors?

Radiotherapy (RT) plays a fundamental role in the treatment of glioblastoma (GBM). GBM are notoriously invasive and harbor a subpopulation of cells with stem-like features which exhibit upregulation of the DNA damage response (DDR) and are radioresistant. High radiation doses are therefore delivered to large brain volumes and are known to extend survival but also cause delayed toxicity with 50%-90% of patients developing neurocognitive dysfunction. Emerging evidence identifies neuroinflammation as a critical mediator of the adverse effects of RT on cognitive function. In addition to its well-established role in promoting repair of radiation-induced DNA damage, activation of poly(ADP-ribose) polymerase (PARP) can exacerbate neuroinflammation by promoting secretion of inflammatory mediators. Therefore, PARP represents an intriguing mechanistic link between radiation-induced activation of the DDR and subsequent neuroinflammation. PARP inhibitors (PARPi) have emerged as promising new agents for GBM when given in combination with RT, with multiple preclinical studies demonstrating radiosensitizing effects and at least 3 compounds being evaluated in clinical trials. We propose that concomitant use of PARPi could reduce radiation-induced neuroinflammation and reduce the severity of radiation-induced cognitive dysfunction while at the same time improving tumor control by enhancing radiosensitivity.

Cytochrome OmcS Is Not Essential for Extracellular Electron Transport via Conductive Pili in Geobacter sulfurreducens Strain KN400.

The multi-heme c-type cytochrome OmcS is one of the central components used for extracellular electron transport in the Geobacter sulfurreducens strain DL-1, but its role in other microbes, including other strains of G. sulfurreducens, is currently a matter of debate. Therefore, we investigated the function of OmcS in the G. sulfurreducens strain KN400, which is even more effective in extracellular electron transfer than the DL-1 strain. We found that deleting omcS from strain KN400 did not negatively impact the rate of Fe(III) oxide reduction and that the cells expressed conductive filaments. Replacing the wild-type pilin gene with the aro-5 pilin gene eliminated the OmcS-deficient strain's ability to transport electrons to insoluble electron acceptors and diminished filament conductivity. These results are consistent with the concept that electrically conductive pili are the primary conduit for long-range electron transfer in G. sulfurreducens and closely related species. These findings, coupled with the lack of OmcS homologs in other microbes capable of extracellular electron transfer, suggest that OmcS is not a common critical component for extracellular electron transfer. IMPORTANCE OmcS has been widely studied and noted to be one of the key components for extracellular electron exchange by the Geobacter sulfurreducens strain DL-1. However, the true importance of OmcS warrants further investigation because it is well known that few bacteria, even within the Geobacteraceae family, contain OmcS homologs, and many bacteria that are capable of extracellular electron transfer lack an abundance of any type of outer surface c-type cytochrome. In addition, there is debate about the importance of OmcS filaments in the mechanism of extracellular electron transport to insoluble electron acceptors by G. sulfurreducens. It has been suggested that filaments comprised of OmcS rather than e-pili are the predominant conductive filaments expressed by G. sulfurreducens. However, the results presented here, along with multiple other sources of evidence, indicate that OmcS filaments cannot be the primary, conductive, protein nanowires expressed by G. sulfurreducens.

Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens.

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90% of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in Escherichia coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90% filaments with a 3-nm diameter, but greatly reduced conductance, further indicating that the 3-nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, were deleted yielded only 3-nm-diameter filaments with the same conductance as in the wild type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer. IMPORTANCE Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport. This study demonstrates that e-pili are abundant filaments emanating from Geobacter sulfurreducens, which serves as a model for long-range extracellular electron transfer in direct interspecies electron transfer, dissimilatory metal reduction, microbe-electrode exchange, and corrosion caused by direct electron uptake from Fe(0). The methods described in this study provide a simple strategy for evaluating the distribution of conductive filaments throughout the microbial world with an approach that avoids artifactual production and/or enrichment of filaments that may not be physiologically relevant.

Generation of High Current Densities in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor.

Geobacter sulfurreducens is commonly employed as a model for the study of extracellular electron transport mechanisms in the Geobacter species. Deletion of pilB, which is known to encode the pilus assembly motor protein for type IV pili in other bacteria, has been proposed as an effective strategy for evaluating the role of electrically conductive pili (e-pili) in G. sulfurreducens extracellular electron transfer. In those studies, the inhibition of e-pili expression associated with pilB deletion was not demonstrated directly but was inferred from the observation that pilB deletion mutants produced lower current densities than wild-type cells. Here, we report that deleting pilB did not diminish current production. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are expressed heterologously from the G. sulfurreducens pilin gene in Escherichia coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing a pilin monomer with a His tag continued to express His tag-labeled filaments when pilB was deleted. These results suggest that a reinterpretation of the results of previous studies on G. sulfurreducens pilB deletion strains may be necessary. IMPORTANCE Geobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes, such as the reduction of Fe(III) oxides in soils and sediments, bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity, direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments, and metal corrosion. Elucidating the phenotypes associated with gene deletions is an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens. The results reported here demonstrate that we cannot replicate the key phenotype reported for a gene deletion that has been central to the development of models for long-range electron transport in G. sulfurreducens.

Resting and postprandial serum bile acid concentrations in dogs with liver disease.

BACKGROUND: Serum bile acids (SBAs) are frequently measured in dogs. However, there is limited data comparing SBAs in different liver diseases diagnosed according to standardized histological criteria. OBJECTIVES: To compare resting and postprandial SBAs, and determine their sensitivity and specificity, for various liver diseases in dogs. ANIMALS: Three hundred and forty-one client-owned dogs with suspected liver disease that had a liver biopsy and SBAs measured. METHODS: Multicenter retrospective study. Cases were classified according to standardized histological criteria. The sensitivity and specificity of resting and postprandial SBAs for the diagnosis of each liver disease, and all liver diseases combined, were calculated. RESULTS: The median resting SBAs were highest in dogs with cirrhosis (98.8 µmol/L; range, 6-135) and congenital circulatory anomalies (CCa; 79.45 µmol/L; 0.3-705). The highest median postprandial concentrations were found in CCa (126 µmol/L; 0-726) and chronic hepatitis (CH; 54.3 µmol/L; 0-260). Using the cut-off value of 10 µmol/L, the highest sensitivities of resting SBAs were recorded in dogs with CCa (87.5%; 95% confidence interval, 76.8-94.4) and CH (81.1%; 71.5-88.6). The sensitivities of postprandial SBAs were the highest in cholangitis (100%; 47.8-100.0) and CCa (91.1%; 78.8-97.5). The specificities of resting and postprandial SBAs for all diseases were 49.3% (37.6-61.1) and 29.7% (15.9-47.0), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Postprandial SBAs are more sensitive but less specific than resting SBAs for the diagnosis of liver disease. There were dogs in all categories of liver disease with resting SBAs <10 and >90 µmol/L. Therefore, careful interpretation of both normal and elevated values is required.

Inhibition of glycolysis and mitochondrial respiration promotes radiosensitisation of neuroblastoma and glioma cells.

BACKGROUND: Neuroblastoma accounts for 7% of paediatric malignancies but is responsible for 15% of all childhood cancer deaths. Despite rigorous treatment involving chemotherapy, surgery, radiotherapy and immunotherapy, the 5-year overall survival rate of high-risk disease remains < 40%, highlighting the need for improved therapy. Since neuroblastoma cells exhibit aberrant metabolism, we determined whether their sensitivity to radiotherapy could be enhanced by drugs affecting cancer cell metabolism. METHODS: Using a panel of neuroblastoma and glioma cells, we determined the radiosensitising effects of inhibitors of glycolysis (2-DG) and mitochondrial function (metformin). Mechanisms underlying radiosensitisation were determined by metabolomic and bioenergetic profiling, flow cytometry and live cell imaging and by evaluating different treatment schedules. RESULTS: The radiosensitising effects of 2-DG were greatly enhanced by combination with the antidiabetic biguanide, metformin. Metabolomic analysis and cellular bioenergetic profiling revealed this combination to elicit severe disruption of key glycolytic and mitochondrial metabolites, causing significant reductions in ATP generation and enhancing radiosensitivity. Combination treatment induced G2/M arrest that persisted for at least 24 h post-irradiation, promoting apoptotic cell death in a large proportion of cells. CONCLUSION: Our findings demonstrate that the radiosensitising effect of 2-DG was significantly enhanced by its combination with metformin. This clearly demonstrates that dual metabolic targeting has potential to improve clinical outcomes in children with high-risk neuroblastoma by overcoming radioresistance.

Cerebral Microangiopathy in Two Dogs with Cutaneous and Renal Glomerular Vasculopathy.

Cutaneous and renal glomerular vasculopathy (CRGV) is an emerging disease in the UK, but its aetiology remains unclear. It is considered a thrombotic microangiopathy (TMA) in which the kidney and skin are the most commonly affected organs. We now document two cases of CRGV with brain lesions, which may have accounted for neurological signs displayed by these animals. The histopathological brain lesions were similar to TMA lesions in humans with thrombotic thrombocytopaenic purpura (TTP) and complement-mediated haemolytic uraemic syndrome (CM-HUS), in which the neurological signs are more associated with TMA than with any systemic disease or electrolyte imbalance. Fibrinoid necrosis in brain arterioles and associated lesions in these dogs were similar to those in human CM-HUS, indicating that the alternative complement pathway may play an important role in the pathophysiology of CRGV.

Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates.

Extracellular electron transfer (EET) pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging because a functional understanding of its EET pathway proteins and their effect on engineering parametrizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures that predictably turn on and turn off EET flux, respectively, in response to isopropyl ß-D-1-thiogalactopyranoside (IPTG). Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

Bioinspired bio-voltage memristors.

Memristive devices are promising candidates to emulate biological computing. However, the typical switching voltages (0.2-2 V) in previously described devices are much higher than the amplitude in biological counterparts. Here we demonstrate a type of diffusive memristor, fabricated from the protein nanowires harvested from the bacterium Geobacter sulfurreducens, that functions at the biological voltages of 40-100 mV. Memristive function at biological voltages is possible because the protein nanowires catalyze metallization. Artificial neurons built from these memristors not only function at biological action potentials (e.g., 100 mV, 1 ms) but also exhibit temporal integration close to that in biological neurons. The potential of using the memristor to directly process biosensing signals is also demonstrated.

An Escherichia coli Chassis for Production of Electrically Conductive Protein Nanowires.

Geobacter sulfurreducens' pilin-based electrically conductive protein nanowires (e-PNs) are a revolutionary electronic material. They offer novel options for electronic sensing applications and have the remarkable ability to harvest electrical energy from atmospheric humidity. However, technical constraints limit mass cultivation and genetic manipulation of G. sulfurreducens. Therefore, we designed a strain of Escherichia coli to express e-PNs by introducing a plasmid that contained an inducible operon with E. coli genes for type IV pili biogenesis machinery and a synthetic gene designed to yield a peptide monomer that could be assembled into e-PNs. The e-PNs expressed in E. coli and harvested with a simple filtration method had the same diameter (3 nm) and conductance as e-PNs expressed in G. sulfurreducens. These results, coupled with the robustness of E. coli for mass cultivation and the extensive E. coli toolbox for genetic manipulation, greatly expand the opportunities for large-scale fabrication of novel e-PNs.

Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option.

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron transfer because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Heterologous expression of the putative S. aciditrophicus type IV pilin gene in Geobacter sulfurreducens yielded conductive pili of the same diameter (4 nm) and conductance of the native S. aciditrophicus pili and enabled long-range electron transport in G. sulfurreducens. S. aciditrophicus lacked abundant c-type cytochromes often associated with DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

Geobacter Protein Nanowires.

The study of electrically conductive protein nanowires in Geobacter sulfurreducens has led to new concepts for long-range extracellular electron transport, as well as for the development of sustainable conductive materials and electronic devices with novel functions. Until recently, electrically conductive pili (e-pili), assembled from the PilA pilin monomer, were the only known Geobacter protein nanowires. However, filaments comprised of the multi-heme c-type cytochrome, OmcS, are present in some preparations of G. sulfurreducens outer-surface proteins. The purpose of this review is to evaluate the available evidence on the in vivo expression of e-pili and OmcS filaments and their biological function. Abundant literature demonstrates that G. sulfurreducens expresses e-pili, which are required for long-range electron transport to Fe (III) oxides and through conductive biofilms. In contrast, there is no definitive evidence yet that wild-type G. sulfurreducens express long filaments of OmcS extending from the cells, and deleting the gene for OmcS actually increases biofilm conductivity. The literature does not support the concern that many previous studies on e-pili were mistakenly studying OmcS filaments. For example, heterologous expression of the aromatic-rich pilin monomer of Geobacter metallireducens in G. sulfurreducens increases the conductivity of individual nanowires more than 5,000-fold, whereas expression of an aromatic-poor pilin reduced conductivity more than 1,000-fold. This more than million-fold range in nanowire conductivity was achieved while maintaining the 3-nm diameter characteristic of e-pili. Purification methods that eliminate all traces of OmcS yield highly conductive e-pili, as does heterologous expression of the e-pilin monomer in microbes that do not produce OmcS or any other outer-surface cytochromes. Future studies of G. sulfurreducens expression of protein nanowires need to be cognizant of the importance of maintaining environmentally relevant growth conditions because artificial laboratory culture conditions can rapidly select against e-pili expression. Principles derived from the study of e-pili have enabled identification of non-cytochrome protein nanowires in diverse bacteria and archaea. A similar search for cytochrome appendages is warranted. Both e-pili and OmcS filaments offer design options for the synthesis of protein-based "green" electronics, which may be the primary driving force for the study of these structures in the near future.

Decorating the Outer Surface of Microbially Produced Protein Nanowires with Peptides.

The potential applications of electrically conductive protein nanowires (e-PNs) harvested from Geobacter sulfurreducens might be greatly expanded if the outer surface of the wires could be modified to confer novel sensing capabilities or to enhance binding to other materials. We developed a simple strategy for functionalizing e-PNs with surface-exposed peptides. The G. sulfurreducens gene for the monomer that assembles into e-PNs was modified to add peptide tags at the carboxyl terminus of the monomer. Strains of G. sulfurreducens were constructed that fabricated synthetic e-PNs with a six-histidine "His-tag" or both the His-tag and a nine-peptide "HA-tag" exposed on the outer surface. Addition of the peptide tags did not diminish e-PN conductivity. The abundance of HA-tag in e-PNs was controlled by placing expression of the gene for the synthetic monomer with the HA-tag under transcriptional regulation. These studies suggest broad possibilities for tailoring e-PN properties for diverse applications.

The Archaellum of Methanospirillum hungatei Is Electrically Conductive.

Microbially produced electrically conductive protein filaments are of interest because they can function as conduits for long-range biological electron transfer. They also show promise as sustainably produced electronic materials. Until now, microbially produced conductive protein filaments have been reported only for bacteria. We report here that the archaellum of Methanospirillum hungatei is electrically conductive. This is the first demonstration that electrically conductive protein filaments have evolved in Archaea Furthermore, the structure of the M. hungatei archaellum was previously determined (N. Poweleit, P. Ge, H. N. Nguyen, R. R. O. Loo, et al., Nat Microbiol 2:16222, 2016, https://doi.org/10.1038/nmicrobiol.2016.222). Thus, the archaellum of M. hungatei is the first microbially produced electrically conductive protein filament for which a structure is known. We analyzed the previously published structure and identified a core of tightly packed phenylalanines that is one likely route for electron conductance. The availability of the M. hungatei archaellum structure is expected to substantially advance mechanistic evaluation of long-range electron transport in microbially produced electrically conductive filaments and to aid in the design of "green" electronic materials that can be microbially produced with renewable feedstocks.IMPORTANCE Microbially produced electrically conductive protein filaments are a revolutionary, sustainably produced, electronic material with broad potential applications. The design of new protein nanowires based on the known M. hungatei archaellum structure could be a major advance over the current empirical design of synthetic protein nanowires from electrically conductive bacterial pili. An understanding of the diversity of outer-surface protein structures capable of electron transfer is important for developing models for microbial electrical communication with other cells and minerals in natural anaerobic environments. Extracellular electron exchange is also essential in engineered environments such as bioelectrochemical devices and anaerobic digesters converting wastes to methane. The finding that the archaellum of M. hungatei is electrically conductive suggests that some archaea might be able to make long-range electrical connections with their external environment.

Early-life adversity programs long-term cytokine and microglia expression within the HPA axis in female Japanese quail.

Stress exposure during prenatal and postnatal development can have persistent and often dysfunctional effects on several physiological systems, including immune function, affecting the ability to combat infection. The neuroimmune response is inextricably linked to the action of the hypothalamic-pituitary-adrenal (HPA) axis. Cytokines released from neuroimmune cells, including microglia, activate the HPA axis, while glucocorticoids in turn regulate cytokine release from microglia. Because of the close links between these two physiological systems, coupled with potential for persistent changes to HPA axis activity following developmental stress, components of the neuroimmune system could be targets for developmental programming. However, little is known of any programming effects of developmental stress on neuroimmune function. We investigated whether developmental stress exposure via elevated prenatal corticosterone (CORT) or postnatal unpredictable food availability had long-term effects on pro- (IL-1ß) and anti-inflammatory (IL-10) cytokine and microglia-dependent gene (CSF1R) expression within HPA axis tissues in a precocial bird, the Japanese quail (Coturnix japonica). Following postnatal stress, we observed increased IL-1ß expression in the pituitary gland, reduced IL-10 expression in the amygdala and hypothalamus, and reduced CSF1R expression within the hypothalamus and pituitary gland. Postnatal stress disrupted the ratio of IL-1ß:IL-10 expression within the hippocampus and hypothalamus. Prenatal stress only increased IL-1ß expression in the pituitary gland. We found no evidence for interactive or cumulative effects across life stages on basal cytokine and glia expression in adulthood. We show that postnatal stress may have a larger impact than elevated prenatal CORT on basal immunity in HPA-axis-specific brain regions, with changes in cytokine homeostasis and microglia abundance. These results provide evidence for postnatal programming of a pro-inflammatory neuroimmune phenotype at the expense of reduced microglia, which could have implications for central nervous system health and subsequent neuroimmune responses.