Kidney organoids: steps towards better organization and function.

Kidney organoids - 3D representations of kidneys made either from pluripotent or tissue stem cells - have been available for well over a decade. Their application could confer notable benefits over longstanding in vivo approaches with the potential for clinically aligned human cells and reduced ethical burdens. They been used, at a proof-of-concept level, in development in disease modeling (including with patient-derived stem cells), and in screening drugs for efficacy/toxicity. They differ from real kidneys: they represent only foetal-stage tissue, in their simplest forms they lack organ-scale anatomical organization, they lack a properly arranged vascular system, and include non-renal cells. Cell specificity may be improved by better techniques for differentiation and/or sorting. Sequential assembly techniques that mimic the sequence of natural development, and localized sources of differentiation-inducing signals, improve organ-scale anatomy. Organotypic vascularization remains a challenge: capillaries are easy, but the large vessels that should serve them are absent from organoids and, even in cultured real kidneys, these large vessels do not survive without blood flow. Transplantation of organoids into hosts results in their being vascularized (though probably not organotypically) and in some renal function. It will be important to transplant more advanced organoids, with a urine exit, in the near future to assess function more stringently. Transplantation of human foetal kidneys, followed by nephrectomy of host kidneys, keeps rats alive for many weeks, raising hope that, if organoids can be produced even to the limited size and complexity of foetal kidneys, they may one day be useful in renal replacement.

Portable gas chromatography-mass spectrometry method for the in-field screening of organic pollutants in soil and water at pollution incidents.

Environmental pollution incidents generate an emergency response from regulatory agencies to ensure that the impact on the environment is minimised. Knowing what pollutants are present provides important intelligence to assist in determining how to respond to the incident. However, responders are limited in their in-field capabilities to identify the pollutants present. This research has developed an in-field, qualitative analytical approach to detect and identify organic pollutants that are commonly detected by regulatory environmental laboratories. A rapid, in-field extraction method was used for water and soil matrices. A coiled microextraction (CME) device was utilised for the introduction of the extracted samples into a portable gas chromatography-mass spectrometry (GC-MS) for analysis. The total combined extraction and analysis time was approximately 6.5 min per sample. Results demonstrated that the in-field extraction and analysis methods can screen for fifty-nine target organic contaminants, including polyaromatic hydrocarbons, monoaromatic hydrocarbons, phenols, phthalates, organophosphorus pesticides, and organochlorine pesticides. The method was also capable of tentatively identifying unknown compounds using library searches, significantly expanding the scope of the methods for the provision of intelligence at pollution incidents of an unknown nature, although a laboratory-based method was able to provide more information due to the higher sensitivity achievable. The methods were evaluated using authentic casework samples and were found to be fit-for-purpose for providing rapid in-field intelligence at pollution incidents. The fact that the in-field methods target the same compounds as the laboratory-based methods provides the added benefit that the in-field results can assist in sample triaging upon submission to the laboratory for quantitation and confirmatory analysis.

Innervation of the developing kidney in vivo and in vitro.

Within the adult kidney, renal neurites can be observed alongside the arteries where they play a role in regulating blood flow. However, their role and localization during development has so far not been described in detail. In other tissues, such as the skin of developing limb buds, neurons play an important role during arterial differentiation. Here, we aim to investigate whether renal nerves could potentially carry out a similar role during arterial development in the mouse kidney. In order to do so, we used whole-mount immunofluorescence staining to identify whether the timing of neuronal innervation correlates with the recruitment of arterial smooth muscle cells. Our results show that neurites innervate the kidney between day 13.5 and 14.5 of development, arriving after the recruitment of smooth muscle actin-positive cells to the renal arteries.

Stratified tissue biofabrication by rotational internal flow layer engineering.

The bioassembly of layered tissue that closely mimics human histology presents challenges for tissue engineering. Existing bioprinting technologies lack the resolution and cell densities necessary to form the microscale cell-width layers commonly observed in stratified tissue, particularly when using low-viscosity hydrogels, such as collagen. Here we present rotational internal flow layer engineering (RIFLE), a novel, low-cost biofabrication technology for assembling tuneable, multi-layered tissue-like structures. Using high-speed rotating tubular moulds, small volumes of cell-laden liquids added to the inner surface were transitioned into thin layers and gelled, progressively building macroscale tubes composed of discrete microscale strata with thicknesses a function of rotational speed. Cell encapsulation enabled the patterning of high-density layers (108cells ml-1) into heterogenous constructs. RIFLE versatility was demonstrated through tunica media assembly, encapsulating human smooth muscle cells in cell-width (12.5µm) collagen layers. Such deposition of discrete microscale layers, facilitates the biofabrication of composite structures mimicking the nature of native stratified tissue. This enabling technology has the potential to allow researchers to economically create a range of representative layered tissue.

Bioactive α,ß-conjugated 3-keto-steroids from the Australian brown alga Cystophora xiphocarpa.

As part of our ongoing study of the specialised metabolites present in brown algae belonging to the Cystophora genus, eight new steroids including three pairs of diastereoisomers were isolated from Cystophora xiphocarpa (Harvey) (Sargassacea, Fucales). The metabolites identified by standard spectrometric methods are (16S,22S)-16,22-dihydroxyergosta-4,24(28)-dien-3-one and (16S,22R)-16,22-dihydroxyergosta-4,24(28)-dien-3-one, (16S,22S,24R)-16,22,24-trihydroxyporifera-4,28-dien-3-one and (16S,22S,24S)-16,22,24-trihydroxystigma-4,28-dien-3-one along with (16S,22S,24E)-16,22-dihydroxystigma-4,24(28)-dien-3-one and (16S,20S)-16,20-dihydroxyergosta-4,24(28)-dien-3-one. (16S,22S,24E)-16,22-Dihydroxystigma-4,24(28)-dien-3-one possessed the most potent cytotoxicity of the steroids in this series with cell growth inhibitions of GI50 8.7 ± 0.7 µM against colon cancer HT29, GI50 5.6 ± 0.8 µM against the breast cancer line MCF-7 and GI50 4.5 ± 0.2 µM against the ovarian cancer cell line A2780. (16S,22R)-16,22-dihydroxyergosta-4,24(28)-dien-3-one was found to be active against the ovarian cancer cell line A2780 with a GI50 of 6.2 ± 0.1 µM.

COVID-19 Outbreaks in Correctional Facilities with Work-Release Programs - Idaho, July-November 2020.

As of April 16, 2021, U.S. correctional and detention facilities reported 399,631 cases of COVID-19 in incarcerated persons, resulting in 2,574 deaths (1). During July 14-November 30, 2020, COVID-19 was diagnosed in 382 persons incarcerated in Idaho correctional facilities with work-release programs. Work-release programs (which place incarcerated persons in community businesses) have social and economic benefits, but might put participants at increased risk for bidirectional transmission of SARS-CoV-2, the virus that causes COVID-19. The Idaho Department of Correction (IDOC) operates 13 state-run correctional facilities, including six low-security facilities dedicated to work-release programs. This report describes COVID-19 outbreaks in five IDOC facilities with work-release programs,* provides the mitigation strategies that IDOC implemented, and describes the collaborative public health response. As of November 30, 2020, 382 outbreak-related COVID-19 cases were identified among incarcerated persons in five Idaho correctional facilities with work-release programs; two outbreaks were linked to food processing plants. Mitigation strategies that helped to control outbreaks in IDOC facilities with work-release programs included isolation of persons with COVID-19, identification and quarantine of close contacts, mass testing of incarcerated persons and staff members, and temporary suspension of work-release programs. Implementation of public health recommendations for correctional and detention facilities with work-release programs, including mass testing and identification of high-risk work sites, can help mitigate SARS-CoV-2 outbreaks. Incarcerated persons participating in work-release should be included in COVID-19 vaccination plans.

Automation in the Life Science Research Laboratory.

Protocols in the academic life science laboratory are heavily reliant on the manual manipulation of tools, reagents and instruments by a host of research staff and students. In contrast to industrial and clinical laboratory environments, the usage of automation to augment or replace manual tasks is limited. Causes of this 'automation gap' are unique to academic research, with rigid short-term funding structures, high levels of protocol variability and a benevolent culture of investment in people over equipment. Automation, however, can bestow multiple benefits through improvements in reproducibility, researcher efficiency, clinical translation, and safety. Less immediately obvious are the accompanying limitations, including obsolescence and an inhibitory effect on the freedom to innovate. Growing the range of automation options suitable for research laboratories will require more flexible, modular and cheaper designs. Academic and commercial developers of automation will increasingly need to design with an environmental awareness and an understanding that large high-tech robotic solutions may not be appropriate for laboratories with constrained financial and spatial resources. To fully exploit the potential of laboratory automation, future generations of scientists will require both engineering and biology skills. Automation in the research laboratory is likely to be an increasingly critical component of future research programs and will continue the trend of combining engineering and science expertise together to answer novel research questions.

3D biofabrication for tubular tissue engineering.

The therapeutic replacement of diseased tubular tissue is hindered by the availability and suitability of current donor, autologous and synthetically derived protheses. Artificially created, tissue engineered, constructs have the potential to alleviate these concerns with reduced autoimmune response, high anatomical accuracy, long-term patency and growth potential. The advent of 3D bioprinting technology has further supplemented the technological toolbox, opening up new biofabrication research opportunities and expanding the therapeutic potential of the field. In this review, we highlight the challenges facing those seeking to create artificial tubular tissue with its associated complex macro- and microscopic architecture. Current biofabrication approaches, including 3D printing techniques, are reviewed and future directions suggested.

Towards non-invasive characterisation of coronary stent re-endothelialisation - An in-vitro, electrical impedance study.

The permanent implantation of a stent has become the most common method for ameliorating coronary artery narrowing arising from atherosclerosis. Following the procedure, optimal arterial wall healing is characterised by the complete regrowth of an Endothelial Cell monolayer over the exposed stent surface and surrounding tissue, thereby reducing the risk of thrombosis. However, excessive proliferation of Smooth Muscle Cells, within the artery wall can lead to unwanted renarrowing of the vessel lumen. Current imaging techniques are unable to adequately identify re-endothelialisation, and it has previously been reported that the stent itself could be used as an electrode in combination with electrical impedance spectroscopic techniques to monitor the post-stenting recovery phase. The utility of such a device will be determined by its ability to characterise between vascular cell types. Here we present in-vitro impedance spectroscopy measurements of pulmonary artery porcine Endothelial Cells, Human Umbilical Vein Endothelial Cells and coronary artery porcine Smooth Muscle Cells grown to confluence over platinum black electrodes in clinically relevant populations. These measurements were obtained, using a bespoke impedance spectroscopy system that autonomously performed impedance sweeps in the 1kHz to 100kHz frequency range. Analysis of the reactance component of impedance revealed distinct frequency dependent profiles for each cell type with post confluence reactance declines in Endothelial Cell populations that have not been previously reported. Such profiles provide a means of non-invasively characterising between the cell types and give an indication that impedance spectroscopic techniques may enable the non-invasive characterisation of the arterial response to stent placement.

Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145.

The Streptomyces genus is well known for its ability to produce bio-active secondary metabolites of great medical interest. However, the metabolic features accompanying these bio-productions remain to be defined. In this study, the comparison of related model strains producing differing levels of actinorhoddin (ACT), showed that S. lividans, a weak producer, had high TriAcylGlycerol (TAG) content indicative of a glycolytic metabolism. In contrast, the strong producer, S. coelicolor, was characterized by low TAG content, active consumption of its polyphosphate (PolyP) stores and extremely high ATP/ADP ratios. This indicated highly active oxidative metabolism that was correlated with induction of ACT biosynthesis. Interestingly, in conditions of phosphate limitation, the ppk mutant had TAG content and ACT production levels intermediary between those of S. lividans and S. coelicolor. This strain was characterized by high ADP levels indicating that Ppk was acting as an Adenosine Di Phosphate Kinase. Its absence resulted in energetic stress that is proposed to trigger an activation of oxidative metabolism to restore its energetic balance. This process, which is correlated with ACT biosynthesis, requires acetylCoA to fuel the Krebs cycle and phosphate for ATP generation by the ATP synthase coupled to the respiratory chain, resulting in low TAG and polyP content of the ACT producing strains.

Phosphate Homeostasis in Conditions of Phosphate Proficiency and Limitation in the Wild Type and the phoP Mutant of Streptomyces lividans.

Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphorylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore, short polyP was shown to be present outside the cell, tightly bound to the mycelium via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.

Cytotoxic compounds from Laurencia pacifica.

BACKGROUND: The current investigation sought to explore the nature of the secondary metabolites in the algae, Laurencia pacifica. RESULTS: This report details the first isolation of the sesquiterpenes isoaplysin (1), isolaurenisol (2), debromoisolaurinterol (3), debromoaplysinol (4), laur-11-en-10-ol (5), 10α-hydroxyldebromoepiaplysin (6), and the previously unknown 10-bromo-3,7,11,11-tetramethylspiro[5.5]undeca-1,7-dien-3-ol (7) from the algae, Laurencia pacifica. Isoaplysin (1) and debromoaplysinol (4) showed promising levels of growth inhibition against a panel cancer-derived cell lines of colon (HT29), glioblastoma (U87, SJ-G2), breast (MCF-7), ovarian (A2780), lung (H460), skin (A431), prostate (Du145), neuroblastoma (BE2-C), pancreas (MIA), murine glioblastoma (SMA) origin with average GI50 values of 23 and 14 µM. CONCLUSIONS: Isoaplysin (1) and debromoaplysinol (4) were up to fourfold more potent in cancer-derived cell populations than in non-tumor-derived normal cells (MCF10A). These analogues are promising candidates for anticancer drug development. Graphical Abstract ᅟ.

Management of white patches.

White patches are a worrying finding for patients, in particular with concern arising about the possibility of the lesions being or becoming cancerous. This paper looks at some of the common white patches that present and relevant considerations in deciding on a management plan for them.

Steroids from an Australian sponge Psammoclema sp.

Investigation of an extract of the Australian marine sponge Psammoclema sp. for dynamin I inhibitory activity led to the isolation of four new trihydroxysterols (1-4) related to aragusterol G. These compounds were largely identified by 1D and 2D NMR spectroscopic methods. While 1 was found to be inactive in the dynamin bioassay, bioassays did reveal that compounds 1-4 inhibited the growth of colorectal, breast, ovarian, and prostate cancer cell lines (GI(50) 5-27 microM). The additional insight that these new compounds give to previous SAR studies is discussed briefly.

The fractured edentulous atrophic mandible--open or closed treatment?

DATA SOURCES: The Cochrane Oral Health Group Trials Register, Cochrane Central Register of Controlled Trials, Medline and Embase were used for searches. The Internet was searched for potentially relevant meta-analyses and non-Cochrane systematic reviews, ongoing clinical trials and published dissertations. Reference lists of identified studies were cross-checked for any potentially relevant clinical trials. There were no language restrictions. STUDY SELECTION: Studies were selected if they were randomised controlled trials involving people over 55 years of age with fractures in the symphysis, parasymphysis, body, angle, ramus, condyle, and coronoid process of atrophic edentulous mandibles in which the fracture was a result of trauma, implant insertion or due to pathological fracture. Any studies that compared methods of management (open or closed reduction or fixation) were selected. DATA EXTRACTION AND SYNTHESIS: Screening of eligible studies was conducted in duplicate and independently by two review authors. It was intended to express results as random-effects models using mean differences for continuous outcomes and risk ratios for dichotomous outcomes with 95% confidence intervals. Heterogeneity was to be investigated including both clinical and methodological factors. RESULTS: No eligible randomised controlled trials were identified. CONCLUSIONS: This review illustrates that there is currently inadequate evidence to support the effectiveness of any single approach, either open or closed, in the management of fractured atrophic edentulous mandibles and that, until high-level evidence is available, treatment decisions should continue to be based on clinician's prior experience. This absence of evidence may in part reflect a certain lack of clarity and the apparent diversity and lack of reliability in some of the traditional and normative predictors of successful outcomes.

Translocation of bacterial proteins--an overview.

Recent progress in the understanding of the nature of the extraordinary variety of protein translocation systems, mainly in Gram negative bacteria, is reviewed. This takes us from the insertion of proteins into the inner membrane via the sophisticated Sec apparatus, the lethal injection of Type III proteins into host cells and on to the beautiful machine that assembles the flagellum. Attempts are made to establish some order, some common principles that might explain the variety and the complexity of some systems. The fundamentals considered are the nature of different transport signals, the nature of translocons (a wide variety of inner membrane types, outer membrane translocons are more conserved), the process of docking to translocons, the role of chaperones and the folding of transported proteins, the energetics of translocation, and prospects for future advances.