Analysis of fate determination of vegetative cells with reduced phycocyanin content in a multicellular cyanobacterium using Raman microscopy.

The one-dimensional multicellular cyanobacterium Anabaena sp. PCC 7120 exhibits two different cell types under nitrogen-deprived conditions. We found that the intensity of the Raman band at 1,629 cm -1 , which is associated with phycocyanin, was higher in undifferentiated cells (vegetative cells) than in differentiated cells (heterocysts). We observed cells whose band intensity at 1,629 cm -1 was statistically lower than that of vegetative cells, and named them "proheterocysts". We found that proheterocysts did not necessarily differentiate, and could divide or revert to being vegetative cells, as defined by having a higher band intensity at 1,629 cm -1 .

Progress in Lewis-Acid-Templated Diels-Alder Reactions.

The synthesis of natural products with complicated architectures often requires the use of segments with functional groups that can be structurally transformed with the desired stereogenic centers. Bicyclic 𝛾-lactones have great potential as a suitable segment for natural product synthesis. However, the stereoselective construction of such functionalized bicyclic 𝛾-lactones is not as straightforward as one might expect. The template-mediated Diels-Alder reaction is one of the most powerful and versatile methods for providing bicyclic 𝛾-lactones with high regioselectivity and stereoselectivity. In this reaction, the diene is linked to the dienophile by a temporary tether, allowing the reaction to proceed efficiently, yielding a product that can be used for natural product synthesis. This review describes some important instances of the template-mediated Diels-Alder reaction and its application to the synthesis of biologically active compounds.

Identification of Azalamellarin N as a Pyroptosis Inhibitor.

Pyroptosis is a form of regulated cell death that promotes inflammation; it attracts much attention because its dysregulation leads to various inflammatory diseases. To help explore the precise mechanisms by which pyroptosis is regulated, in this study, we searched for chemical compounds that inhibit pyroptosis. From our original compound library, we identified azalamellarin N (AZL-N), a hexacyclic pyrrole alkaloid, as an inhibitor of pyroptosis induced by R837 (also called imiquimod), which is an agonist of the intracellular multiprotein complex nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. However, whereas the effect of AZL-N on R837-induced pyroptosis was relatively weak, AZL-N strongly inhibited pyroptosis induced by extracellular ATP or nigericin, which are different types of NLRP3 inflammasome agonists. This was in contrast with the results that MCC950, a well-established NLRP3 inhibitor, consistently inhibited pyroptosis irrespective of the type of stimulus. We also found that AZL-N inhibited activation of caspase-1 and apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASC), which are components of the NLRP3 inflammasome. Analysis of the structure-activity relationship revealed that a lactam ring of AZL-N, which has been shown to contribute to the strong binding of AZL-N to its known target protein kinases, is required for its inhibitory effects on pyroptosis. These results suggest that AZL-N inhibits pyroptosis by targeting molecule(s), which may be protein kinase(s), that act upstream of NLRP3 inflammasome activation, rather than by directly targeting the components of the NLRP3 inflammasome. Further identification and analysis of target molecule(s) of AZL-N will shed light on the regulatory mechanisms of pyroptosis, particularly those depending on proinflammatory stimuli.

Draft genome sequence of the basidiomycetous yeast Mrakia hoshinonis JCM 32575T isolated from Ellesmere Island, Canadian High Arctic.

Mrakia hoshinonis JCM 32575 was isolated from glacial sediments on Ellesmere Island in the Canadian High Arctic and described as a new basidiomycetous yeast. This species does not require amino acids and vitamins for growth and can grow at sub-zero temperatures. Here, we report a draft genome sequence of this strain.

Engineered IL-7 synergizes with IL-12 immunotherapy to prevent T cell exhaustion and promote memory without exacerbating toxicity.

Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)-resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.

An engineered immunocytokine with collagen affinity improves the tumor bioavailability, tolerability, and therapeutic efficacy of IL-2.

The clinical utility of human interleukin-2 (hIL-2) is limited by its short serum half-life, preferential activation of regulatory T (TReg) over immune effector cells, and dose-limiting toxicities. We previously engineered F10 immunocytokine (IC), an intramolecularly assembled cytokine/antibody fusion protein that linked hIL-2 to an anti-IL-2 antibody (denoted F10) that extended IL-2 half-life and augmented the immune effector to TReg ratio. Here, we leveraged molecular engineering to improve the anti-tumor therapeutic efficacy and tolerability of F10 IC by developing an iteration, denoted F10 IC-CBD (collagen binding domain), designed for intratumoral administration and in situ retention based on collagen affinity. F10 IC-CBD retained IL-2 bioactivity exclusively in the tumor and eliminated IL-2-associated toxicities. Furthermore, F10 IC exhibited potent single-agent therapeutic efficacy and synergy with systemic immune checkpoint blockade and elicited an abscopal response in mouse tumors models. This engineered fusion protein presents a prototype for the design of intratumoral therapies.

Topically-applied collagen-binding serum albumin-fused interleukin-4 modulates wound microenvironment in non-healing wounds.

Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.

Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

The clinical terrain of immunotherapies in heterogeneous pancreatic cancer: unravelling challenges and opportunities.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer and has abysmal survival rates. In the past two decades, immunotherapeutic agents with success in other cancer types have gradually been trialled against PDACs at different stages of cancer progression, either as a monotherapy or in combination with chemotherapy. Unfortunately, to this day, chemotherapy still prolongs the survival rates the most and is prescribed in clinics despite the severe side effects in other cancer types. The low success rates of immunotherapy against PDAC have been attributed most frequently to its complex and multi-faceted tumour microenvironment (TME) and low mutational burden. In this review, we give a comprehensive overview of the immunotherapies tested in PDAC clinical trials thus far, their limitations, and potential explanations for their failure. We also discuss the existing classification of heterogenous PDACs into cancer, cancer-associated fibroblast, and immune subtypes and their potential opportunity in patient selection as a form of personalisation of PDAC immunotherapy. © 2023 The Pathological Society of Great Britain and Ireland.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

Capillary-Scale Hydrogel Microchannel Networks by Wire Templating.

Microvascular networks are essential for the efficient transport of nutrients, waste products, and drugs throughout the body. Wire-templating is an accessible method for generating laboratory models of these blood vessel networks, but it has difficulty fabricating microchannels with diameters of ten microns and narrower, a requirement for modeling human capillaries. This study describes a suite of surface modification techniques to  selectively control the interactions amongst wires, hydrogels, and world-to-chip interfaces. This wire templating method enables the fabrication of perfusable hydrogel-based rounded cross-section capillary-scale networks whose diameters controllably narrow at bifurcations down to 6.1 ± 0.3 microns in diameter. Due to its low cost, accessibility, and compatibility with a wide range of common hydrogels of tunable stiffnesses such as collagen, this technique may increase the fidelity of experimental models of capillary networks for the study of human health and disease.

Raman spectral analysis of microbial pigment compositions in vegetative cells and heterocysts of multicellular cyanobacterium.

The one-dimensional multicellular cyanobacterium, Anabaena sp. PCC 7120, exhibits a simple topology consisting of two types of cells under the nitrogen-depleted conditions. Although the differentiated (heterocyst) and undifferentiated cells (vegetative cells) were distinguished by their cellular shapes, we found that their internal states, that is, microbial pigment compositions, were distinguished by using a Raman microscope. Almost of Raman bands of the cellular components were assigned to vibrations of the pigments; chlorophyll a, ß-carotene, phycocyanin, and allophycocyanin. We found that the Raman spectral measurement can detect the decomposition of both phycocyanin and allophycocyanin, which are components of the light-harvesting phycobilisome complex in the photosystem II. We observed that the Raman bands of phycocyanin and allophycocyanin exhibited more remarkable decrease in the heterocysts when compared to those of chlorophyll a and ß-carotene. This result indicated the prior decomposition of phycobilisome in the heterocysts. We show that the Raman measurement is useful to detect the change of pigment composition in the cell differentiation.

Imprinting Spatial Helicity Structure of Vector Vortex Beam on Spin Texture in Semiconductors.

We present the transfer of the spatially variant polarization of topologically structured light to the spatial spin texture in a semiconductor quantum well. The electron spin texture, which is a circular pattern with repeating spin-up and spin-down states whose repetition rate is determined by the topological charge, is directly excited by a vector vortex beam with a spatial helicity structure. The generated spin texture efficiently evolves into a helical spin wave pattern owing to the spin-orbit effective magnetic fields in the persistent spin helix state by controlling the spatial wave number of the excited spin mode. By tuning the repetition length and azimuthal angle, we simultaneously generate helical spin waves with opposite phases by a single beam.

Targeting advanced prostate cancer with STEAP1 chimeric antigen receptor T cell and tumor-localized IL-12 immunotherapy.

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen for therapeutic targeting in prostate cancer. Here, we report broad expression of STEAP1 relative to prostate-specific membrane antigen (PSMA) in lethal metastatic prostate cancers and the development of a STEAP1-directed chimeric antigen receptor (CAR) T cell therapy. STEAP1 CAR T cells demonstrate reactivity in low antigen density, antitumor activity across metastatic prostate cancer models, and safety in a human STEAP1 knock-in mouse model. STEAP1 antigen escape is a recurrent mechanism of treatment resistance and is associated with diminished tumor antigen processing and presentation. The application of tumor-localized interleukin-12 (IL-12) therapy in the form of a collagen binding domain (CBD)-IL-12 fusion protein combined with STEAP1 CAR T cell therapy enhances antitumor efficacy by remodeling the immunologically cold tumor microenvironment of prostate cancer and combating STEAP1 antigen escape through the engagement of host immunity and epitope spreading.

Dielectrophoresis-Based Selective Droplet Extraction Microfluidic Device for Single-Cell Analysis.

We developed a microfluidic device that enables selective droplet extraction from multiple droplet-trapping pockets based on dielectrophoresis. The device consists of a main microchannel, five droplet-trapping pockets with side channels, and drive electrode pairs appropriately located around the trapping pockets. Agarose droplets capable of encapsulating biological samples were successfully trapped in the trapping pockets due to the difference in flow resistance between the main and side channels. Target droplets were selectively extracted from the pockets by the dielectrophoretic force generated between the electrodes under an applied voltage of 500 V. During their extraction from the trapping pockets, the droplets and their contents were exposed to an electric field for 400-800 ms. To evaluate whether the applied voltage could potentially damage the biological samples, the growth rates of Escherichia coli cells in the droplets, with and without a voltage applied, were compared. No significant difference in the growth rate was observed. The developed device enables the screening of encapsulated single cells and the selective extraction of target droplets.

Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumor microenvironments. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies, which are being harnessed to improve solid tumor CAR T cell therapies. Here, we describe fully optimized CAR T cells targeting tumor-associated glycoprotein-72 (TAG72) for the treatment of solid tumors, identifying the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. These findings have culminated into a phase 1 trial evaluating safety, feasibility, and bioactivity of TAG72-CAR T cells for the treatment of patients with advanced ovarian cancer ( NCT05225363 ). Preclinically, we found that CAR T cell-mediated IFNγ production facilitated by IL-12 signaling was required for tumor cell killing, which was recapitulated by expressing an optimized membrane-bound IL-12 (mbIL12) molecule on CAR T cells. Critically, mbIL12 cell surface expression and downstream signaling was induced and sustained only following CAR T cell activation. CAR T cells with mbIL12 demonstrated improved antigen-dependent T cell proliferation and potent cytotoxicity in recursive tumor cell killing assays in vitro and showed robust in vivo anti-tumor efficacy in human xenograft models of ovarian cancer peritoneal metastasis. Further, locoregional administration of TAG72-CAR T cells with antigen-dependent IL-12 signaling promoted durable anti-tumor responses against both regional and systemic disease in mice and was associated with improved systemic T cell persistence. Our study features a clinically-applicable strategy to improve the overall efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting both regional and systemic disease.

Genome Sequence of Basidiomycetous Yeast Mrakia gelida MGH-2, Isolated from Skarvsnes Ice-Free Area, East Antarctica.

Basidiomycetous yeast Mrakia gelida MGH-2 has been reported from Surikogi Ike in the Skarvsnes ice-free area, East Antarctica. Here, we report on the high-quality genome sequence of the Mrakia gelida MGH-2 strain analyzed by PacBio Sequel and HiSeq 2500 instruments.

Position-dependent changes in phycobilisome abundance in multicellular cyanobacterial filaments revealed by Raman spectral analysis.

The one-dimensional filamentous cyanobacterium, Anabaena sp. PCC 7120, shows a simple morphological pattern consisting of two distinct cell types under nitrogen-deprived conditions. We found that microbial pigment composition in differentiated (heterocyst) and undifferentiated cells (vegetative cells) can be distinguished using Raman microscopy. The Raman bands associated with phycocyanin and allophycocyanin were of higher intensity in vegetative cells than those in heterocysts. However, these bands had statistically lower intensity in vegetative cells located further away from heterocysts. That is, the pigment composition in individual cells is affected by locational information in a filament.

High-Quality Genome Sequence of Cystobasidium tubakii JCM 31526T, Isolated from East Ongul Island, Antarctica.

Cystobasidium tubakii has been reported as a new basidiomycetous yeast species from East Ongul Island, East Antarctica. This species does not require amino acids and vitamins for growth and can grow at subzero temperatures. Here, we report a high-quality genome sequence of Cystobasidium tubakii strain JCM 31526T, which was isolated from East Antarctica.