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Background Traditional constraints specify that 700 cc of liver should be spared a hepatotoxic dose when delivering liver-directed radiotherapy to reduce the risk of inducing liver failure. We investigated the role of single-photon emission computed tomography (SPECT) to identify and preferentially avoid functional liver during liver-directed radiation treatment planning in patients with preserved liver function but limited functional liver volume after receiving prior hepatotoxic chemotherapy or surgical resection. Methods This phase I trial with a 3 + 3 design evaluated the safety of liver-directed radiotherapy using escalating functional liver radiation dose constraints in patients with liver metastases. Dose-limiting toxicities were assessed 6-8 weeks and 6 months after completing radiotherapy. Results All 12 patients had colorectal liver metastases and received prior hepatotoxic chemotherapy; 8 patients underwent prior liver resection. Median computed tomography anatomical nontumor liver volume was 1584 cc (range = 764-2699 cc). Median SPECT functional liver volume was 1117 cc (range = 570-1928 cc). Median nontarget computed tomography and SPECT liver volumes below the volumetric dose constraint were 997 cc (range = 544-1576 cc) and 684 cc (range = 429-1244 cc), respectively. The prescription dose was 67.5-75 Gy in 15 fractions or 75-100 Gy in 25 fractions. No dose-limiting toxicities were observed during follow-up. One-year in-field control was 57%. One-year overall survival was 73%. Conclusion Liver-directed radiotherapy can be safely delivered to high doses when incorporating functional SPECT into the radiation treatment planning process, which may enable sparing of lower volumes of liver than traditionally accepted in patients with preserved liver function. Trial registration NCT02626312. [ABSTRACT FROM AUTHOR]

Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin-KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin-KO motor cortex indicated significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsfla. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results show that activation of the TNF receptor (TNFR1) NF[kappa]B pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in [SOD1.sup.G93A] astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in [SOD1.sup.G93A] mice significantly prolonged mouse survival. Our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potential in ALS therapy., Introduction Glutamate is an essential excitatory neurotransmitter and, at concentrations of approximately 6 [micro]mol/g (wet weight) in human brain, is the most abundant free amino acid species in the central [...]

The sequences surrounding the -12/-24 motif of the m-xylene-responsive [[sigma].sup.54] promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH [[sigma].sup.54] promoter variants known to have various degrees of efficacy and affinity for [[sigma].sup.54]-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XyIR) fused to the -12/-24 region of the wild-type PnifH and its higher [[sigma].sup.54]-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XyIR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.

Background: The use of hypoallergenic derivatives is considered beneficial to promote the safety and efficacy of allergen-specific immunotherapy. We aimed to assess the efficacy of reduced and alkylated (R/A) Pru p 3, a hypoallergenic folding variant of the major peach allergen, in subcutaneous immunotherapy (SCIT) using a murine model of peach allergy.Methods and Results: After sensitization with Pru p 3, BALB/c mice received SCIT with Pru p 3 or R/A Pru p 3 and were challenged with Pru p 3. SCIT with Pru p 3, but not with R/A Pru p 3, suppressed anaphylaxis upon the challenge significantly. SCIT with Pru p 3 did not suppress Pru p 3-specific IgE and IgG1 production, but enhanced IgG2a production. In contrast, SCIT with R/A Pru p 3 suppressed IgE and IgG1 production, but enhanced IgG2a production only moderately. The therapeutic efficacy of SCIT with Pru p 3 was associated with induction of IL-10 and IFN-γ.Conclusion: Hypoallergenic folding variant of Pru p 3 is not likely an efficacious therapeutic component in SCIT of peach allergy. The lower efficacy of R/A Pru p 3 might be attributed to poor antigenicity and/or weak stability due to its unfolded conformation. [ABSTRACT FROM AUTHOR]

Freeform fabrication has emerged as a key direction in printing biologically-relevant materials and structures. With this emerging technology, complex structures with microscale resolution can be created in arbitrary geometries and without the limitations found in traditional bottom-up or top-down additive manufacturing methods. Recent advances in freeform printing have used the physical properties of microparticle-based granular gels as a medium for the submerged extrusion of bioinks. However, most of these techniques require post-processing or crosslinking for the removal of the printed structures (Miller et al., 2015; Jin et al., 2016) [1,2] . In this communication, we introduce a novel method for the one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite) and polyethylene glycol (PEG). Silk fibroin has been used as a biopolymer for bioprinting in several contexts, but chemical or enzymatic additives or bulking agents are needed to stabilize 3D structures. Our method requires no post-processing of printed structures and allows for in situ physical crosslinking of pure aqueous silk fibroin into arbitrary geometries produced through freeform 3D printing. Statement of Significance 3D bioprinting has emerged as a technology that can produce biologically relevant structures in defined geometries with microscale resolution. Techniques for fabrication of free-standing structures by printing into granular gel media has been demonstrated previously, however, these methods require crosslinking agents and post-processing steps on printed structures. Our method utilizes one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite), with no need for additional crosslinking compounds or post processing of the material. This new method allows for in situ physical crosslinking of pure aqueous silk fibroin into defined geometries produced through freeform 3D printing. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Studies the relation between the physiological control of the sigma[sup 54]-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida. Stringent response mediated by the alarmone (p)ppGpp; Improvement in Pu performance with the overexpression of relA; Direct stimulation of the sigma[sup 54]-dependent transcriptional machinery of Pu by ppGpp.

Pru p 3 is the major peach allergen in the Mediterranean area. It frequently elicits severe reactions, limiting its study in humans, raising the need for animal models to investigate the immunological mechanisms involved. However, no anaphylaxis model exists for Pru p 3. We aimed to develop a model of peach anaphylaxis by sensitising mice with Pru p 3 in combination with lipopolysaccharide (LPS) as an adjuvant. Four groups of mice were sensitised intranasally: untreated; treated with Pru p 3; treated with LPS; treated with Pru p 3 + LPS. After sensitisation mice were intraperitoneally challenged with Pru p 3 and in vivo and in vitro parameters were evaluated. Only mice in the Pru p 3 + LPS group showed anaphylaxis symptoms, including a decrease in temperature. Determination of in vitro parameters showed a Th2 response with an increase of Pru p 3-specific IgE and IgG1. Moreover, at the cellular level, we found increased levels of IgE and IgG1 secreting Pru p 3-specific cells and a proliferative CD4+ T-cell response. These results demonstrate that Pru p 3-specific anaphylaxis can be generated after nasal sensitisation to Pru p 3 in combination with LPS. This is a promising model for evaluating food allergy immunotherapies. [ABSTRACT FROM AUTHOR]

Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin KO motor cortex indicated significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsf1a. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results shown that activation of TNF receptor (TNFR1)-NFκB pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in SOD1G93A astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in SOD1G93A mice significantly prolonged mouse survival. Together, our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potentials in ALS therapy. [ABSTRACT FROM AUTHOR]

Cholesterol metabolism operates autonomously within the central nervous system (CNS), where the majority of cholesterol resides in myelin. We demonstrate that TDP-43, the pathological signature protein for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), influences cholesterol metabolism in oligodendrocytes. TDP-43 binds directly to mRNA of SREBF2, the master transcription regulator for cholesterol metabolism, and multiple mRNAs encoding proteins responsible for cholesterol biosynthesis and uptake, including HMGCR, HMGCS1, and LDLR. TDP-43 depletion leads to reduced SREBF2 and LDLR expression, and cholesterol levels in vitro and in vivo. TDP-43-mediated changes in cholesterol levels can be restored by reintroducing SREBF2 or LDLR. Additionally, cholesterol supplementation rescues demyelination caused by TDP-43 deletion. Furthermore, oligodendrocytes harboring TDP-43 pathology from FTD patients show reduced HMGCR and HMGCS1, and coaggregation of LDLR and TDP-43. Collectively, our results indicate that TDP-43 plays a role in cholesterol homeostasis in oligodendrocytes, and cholesterol dysmetabolism may be implicated in TDP-43 proteinopathies-related diseases., (© 2021 Ho et al.)