Designed modular protein hydrogels for biofabrication.

Designing proteins that fold and assemble over different length scales provides a way to tailor the mechanical properties and biological performance of hydrogels. In this study, we designed modular proteins that self-assemble into fibrillar networks and, as a result, form hydrogel materials with novel properties. We incorporated distinct functionalities by connecting separate self-assembling (A block) and cell-binding (B block) domains into single macromolecules. The number of self-assembling domains affects the rigidity of the fibers and the final storage modulus G' of the materials. The mechanical properties of the hydrogels could be tuned over a broad range (G' = 0.1 - 10 kPa), making them suitable for the cultivation and differentiation of multiple cell types, including cortical neurons and human mesenchymal stem cells. Moreover, we confirmed the bioavailability of cell attachment domains in the hydrogels that can be further tailored for specific cell types or other biological applications. Finally, we demonstrate the versatility of the designed proteins for application in biofabrication as 3D scaffolds that support cell growth and guide their function. STATEMENT OF SIGNIFICANCE: Designed proteins that enable the decoupling of biophysical and biochemical properties within the final material could enable modular biomaterial engineering. In this context, we present a designed modular protein platform that integrates self-assembling domains (A blocks) and cell-binding domains (B blocks) within a single biopolymer. The linking of assembly domains and cell-binding domains this way provided independent tuning of mechanical properties and inclusion of biofunctional domains. We demonstrate the use of this platform for biofabrication, including neural cell culture and 3D printing of scaffolds for mesenchymal stem cell culture and differentiation. Overall, this work highlights how informed design of biopolymer sequences can enable the modular design of protein-based hydrogels with independently tunable biophysical and biochemical properties.

Measuring intraoperative anesthetic parameters during hepatectomy with inferior vena cava clamping.

PURPOSE: Uncontrollable bleeding remained problematic in anatomical hepatectomy exposing hepatic veins. Based on the inferior vena cava (IVC) anatomy, we attempted to analyze the hemodynamic and surgical effects of the combined IVC-partial clamp (PC) accompanied with the Trendelenburg position (TP). METHODS: We prospectively assessed 26 consecutive patients who underwent anatomical hepatectomies exposing HV trunks between 2020 and 2023. Patients were divided into three groups: use of IVC-PC (group 1), no use of IVC-PC (group 2), and use of IVC-PC accompanied with TP (group 3). In 10 of 26 patients (38%), hepatic venous pressure was examined using transhepatic catheter insertion. RESULTS: IVC-PC was performed in 15 patients (58%). Operating time and procedures did not significantly differ between groups. A direct hemostatic effect on hepatic veins was evaluated in 60% and 70% of patients in groups 1 and 3, respectively. Group 1 showed significantly more unstable vital status and vasopressor use (p < 0.01). Blood or fluid transfusion and urinary output were similar between groups. Group 2 had a significantly lower baseline central venous pressure (CVP), while group 3 showed a significant increase in CVP in TP. CVP under IVC-PC seemed lower than under TP; however, not significantly. Hepatic venous pressure did not significantly differ between groups. Systolic arterial blood pressure significantly decreased via IVC-PC in group 1 and to a similar extent in group 3. Heart rate significantly increased during IVC-PC (p < 0.05). CONCLUSION: IVC-PC combined with the TP may be an alternative procedure to control intrahepatic venous bleeding during anatomical hepatectomy exposing hepatic venous trunks.

Regulation of the Ca2+ Channel CaV1.2 Supports Spatial Memory and Its Flexibility and LTD.

Widespread release of norepinephrine (NE) throughout the forebrain fosters learning and memory via adrenergic receptor (AR) signaling, but the molecular mechanisms are largely unknown. The ß2 AR and its downstream effectors, the trimeric stimulatory Gs-protein, adenylyl cyclase (AC), and the cAMP-dependent protein kinase A (PKA), form a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. Phosphorylation of CaV1.2 by PKA on Ser1928 is required for the upregulation of Ca2+ influx on ß2 AR stimulation and long-term potentiation induced by prolonged theta-tetanus (PTT-LTP) but not LTP induced by two 1-s-long 100-Hz tetani. However, the function of Ser1928 phosphorylation in vivo is unknown. Here, we show that S1928A knock-in (KI) mice of both sexes, which lack PTT-LTP, express deficiencies during initial consolidation of spatial memory. Especially striking is the effect of this mutation on cognitive flexibility as tested by reversal learning. Mechanistically, long-term depression (LTD) has been implicated in reversal learning. It is abrogated in male and female S1928A knock-in mice and by ß2 AR antagonists and peptides that displace ß2 AR from CaV1.2. This work identifies CaV1.2 as a critical molecular locus that regulates synaptic plasticity, spatial memory and its reversal, and LTD.SIGNIFICANCE STATEMENT We show that phosphorylation of the Ca2+ channel CaV1.2 on Ser1928 is important for consolidation of spatial memory and especially its reversal, and long-term depression (LTD). Identification of Ser1928 as critical for LTD and reversal learning supports the model that LTD underlies flexibility of reference memory.

A phase III, multicenter, single-arm study to assess the utility of indocyanine green fluorescent lymphography in the treatment of secondary lymphedema.

OBJECTIVE: Indocyanine green (ICG) fluorescent lymphography might be useful for assessing patients undergoing lymphatic surgery for secondary lymphedema. The present clinical trial aimed to confirm whether ICG fluorescent lymphography would be useful in evaluating lymphedema, identifying lymphatic vessels suitable for anastomosis, and confirming patency of lymphaticovenular anastomosis in patients with secondary lymphedema. METHODS: The present phase III, multicenter, single-arm, open-label, clinical trial (HAMAMATSU-ICG study) investigated the accuracy of lymphedema diagnosis via ICG fluorescent lymphography compared with lymphoscintigraphy, rate of identification of lymphatic vessels at the incision site, and efficacy for confirming patency of lymphaticovenular anastomosis. The external diameter of the identified lymphatic vessels and the distance from the skin surface to the lymphatic vessels using preoperative ICG fluorescent lymphography were measured intraoperatively under surgical microscopy. RESULTS: When the clinical decision for surgery at each research site was made, the standard diagnosis of lymphedema was considered correct. For the 26 upper extremities, a central judgment committee who was unaware of the clinical presentation confirmed the imaging diagnosis was accurate for 100.0% of cases, whether the assessments had been performed via lymphoscintigraphy or ICG lymphography. In contrast, for the 88 lower extremities, the accuracy of the diagnosis compared with the diagnosis by the central judgment committee was 70.5% and 88.2% for lymphoscintigraphy and ICG lymphography, respectively. The external diameter of the identified lymphatic vessels was significantly greater in the lower extremities than in the upper extremities (0.54 ± 0.21 mm vs 0.42 ± 0.14 mm; P < .0001). Also, the distance from the skin surface to the lymphatic vessels was significantly longer in the lower extremities than in the upper extremities (5.8 ± 3.5 mm vs 4.4 ± 2.6 mm; P = .01). For 263 skin incisions, with the site placement determined using ICG fluorescent lymphography, the rate of identification of lymphatics vessels suitable for anastomosis was 97.7% (95% confidence interval, 95.1%-99.2%). A total of 267 lymphaticovenular anastomoses were performed. ICG fluorescent lymphography was judged as "useful" for confirming patency after the anastomosis in 95.1% of the cases. CONCLUSIONS: ICG fluorescent lymphography could be useful for improving the treatment of patients with secondary lymphedema from the outpatient setting to surgery.

p53 plays a crucial role in endothelial dysfunction associated with hyperglycemia and ischemia.

p53 is a guardian of the genome that protects against carcinogenesis. There is accumulating evidence that p53 is activated with aging. Such activation has been reported to contribute to various age-associated pathologies, but its role in vascular dysfunction is largely unknown. The aim of this study was to investigate whether activation of endothelial p53 has a pathological effect in relation to endothelial function. We established endothelial p53 loss-of-function and gain-of-function models by breeding endothelial-cell specific Cre mice with floxed Trp53 or floxed Mdm2/Mdm4 mice, respectively. Then we induced diabetes by injection of streptozotocin. In the diabetic state, endothelial p53 expression was markedly up-regulated and endothelium-dependent vasodilatation was significantly impaired. Impairment of vasodilatation was significantly ameliorated in endothelial p53 knockout (EC-p53 KO) mice, and deletion of endothelial p53 also significantly enhanced the induction of angiogenesis by ischemia. Conversely, activation of endothelial p53 by deleting Mdm2/Mdm4 reduced both endothelium-dependent vasodilatation and ischemia-induced angiogenesis. Introduction of p53 into human endothelial cells up-regulated the expression of phosphatase and tensin homolog (PTEN), thereby reducing phospho-eNOS levels. Consistent with these results, the beneficial impact of endothelial p53 deletion on endothelial function was attenuated in EC-p53 KO mice with an eNOS-deficient background. These results show that endothelial p53 negatively regulates endothelium-dependent vasodilatation and ischemia-induced angiogenesis, suggesting that inhibition of endothelial p53 could be a novel therapeutic target in patients with metabolic disorders.

Induction of specific neuron types by overexpression of single transcription factors.

Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analysis of data on gene expression changes 2 d after induction of each of 185 TFs implicated candidate TFs for further ESC differentiation studies. Induction of 23 TFs (out of 49 TFs tested) for 6 d facilitated neural differentiation of ESCs as inferred from increased proportion of cells with neural progenitor marker PSA-NCAM. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In further studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. In summary, this study indicates that induction of transcription factors is a promising approach to generate cultures that show the transcription profiles characteristic of specific neural cell types.

Crystalline Isotactic Polar Polypropylene from the Palladium-Catalyzed Copolymerization of Propylene and Polar Monomers.

Moderately isospecific homopolymerization of propylene and the copolymerization of propylene and polar monomers have been achieved with palladium complexes bearing a phosphine-sulfonate ligand. Optimization of substituents on the phosphorus atom of the ligand revealed that the presence of bulky alkyl groups (e.g. menthyl) is crucial for the generation of high-molecular-weight polypropylenes (Mw ≈10(4) ), and the substituent at the ortho-position relative to the sulfonate group influences the molecular weight and isotactic regularity of the obtained polypropylenes. Statistical analysis suggested that the introduction of substituents at the ortho-position relative to the sulfonate group favors enantiomorphic site control over chain end control in the chain propagation step. The triad isotacticity could be increased to mm=0.55-0.59, with formation of crystalline polar polypropylenes, as supported by the presence of melting points and sharp peaks in the corresponding X-ray diffraction patterns.

Quantification of the steric influence of alkylphosphine-sulfonate ligands on polymerization, leading to high-molecular-weight copolymers of ethylene and polar monomers.

A series of palladium/alkylphosphine-sulfonate catalysts were synthesized and examined in the homopolymerization of ethylene and the copolymerization of ethylene and polar monomers. Catalysts with alkylphosphine-sulfonate ligands containing sterically demanding alkyl substituents afforded (co)polymers whose molecular weight was increased by up to 2 orders of magnitude relative to polymers obtained from previously reported catalyst systems. The polymer molecular weight was found to be closely correlated to the Sterimol B5 parameter of the alkyl substituents in the alkylphosphine-sulfonate ligands. Thus, the use of bulky alkylphosphine-sulfonate ligands represents an effective and versatile method to prepare high-molecular-weight copolymers of ethylene and various polar monomers, which are difficult to obtain by previously reported methods.

Ethylene/allyl monomer cooligomerization by nickel/phosphine-sulfonate catalysts.

Nickel(II) complexes bearing a phosphine-sulfonate ligand, [(R(2)PC(6)H(4)SO(3))NiMe(2,6-lutidine)] (R = cyclohexyl, 2-MeOC(6)H(4), and 2-[2',6'-(MeO)(2)C(6)H(3)]C(6)H(4), were synthesized and applied as catalysts for the cooligomerizations of ethylene and allyl monomers.

Deep skin infection of Scedosporium apiospermum in a patient with refractory idiopathic thrombocytopenic purpura.

Infection of Scedosporium apiospermum is very rare but is now emerging as an important cause of both localized and disseminated infections in immunocompromised patients. A 62-year-old woman, who had undergone steroid therapy for refractory idiopathic thrombocytopenic purpura and had a history of diffuse large B cell lymphoma, developed a deep skin ulcer complicated with lymphangitis. After culture study demonstrated the presence of S. apiospermum, voriconazole (VRCZ) was administered and prompt improvement was observed. Because it is difficult to distinguish S. apiospermum from Aspergillus by histopathology and S. apiospermum is resistant to amphotericin B, VRCZ should be selected as the first choice of antifungal agent when mold is considered to be the causative organism.

Radiological follow-up study of rheumatoid wrists after radio-lunate limited arthrodesis with ulnar head resection.

Radio-lunate limited arthrodesis (RLA) is an established surgical intervention for damaged rheumatoid arthritis (RA) wrists. The goal of RLA is to conserve the range of motion (ROM) and level of activities of daily life (ADL) by delaying more extensive fusion. In this study, we retrospectively reviewed the outcome of RLA with ulnar head resection (UHR) in terms of pain score, ROM, and radiographic parameters, including carpal height ratio, ulnar translation ratio, and palmar subluxation. The clinical factors, such as dose of corticosteroids per day and laboratory examination data before surgery, including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and rheumatoid factor (RF), did not influence the results of the surgery. The changes in these clinical factors were also statistically analyzed in relation to the Schulthess classification of preoperative radiographs of wrists reported by Simmen and Huber: Type I (ankylosis type), Type II (osteoarthritis type), and Type III (disintegration type). This study hypothesized that RLA with UHR would provide no significant improvement in Type III wrists. The results showed that RLA with UHR resulted in stable wrists with excellent pain relief and with limited, but temporary, functional ROM, suggesting that this procedure seems to be applicable for all types of wrists within this group of patients.

[Two cases of rheumatoid arthritis developed after polymyositis].

We report two cases of rheumatoid arthritis (RA) who later had developed after polymyositis (PM). The first patient was 64-year old male who experienced muscular weakness of the four limbs in proximity 10 years ago. He was diagnosed as PM because of the elevated serum CK and the myogenic pattern of EMG, and his symptoms were improved by treatment with corticosteroid. He started to complain polyarthralgia 2 years ago, followed by interstitial pneumonia, pleuritis and skin ulcer. He was admitted because of exacerbated polyarthralgia, multiple subcutaneous nodules, skin eruption and fever. The level of serum CK was within normal range but CRP was elevated and CH 50 was decreased. The laboratory examination showed positive cryoglobulin and high titer of rheumatoid factor, but anti-Jo 1 antibody was negative. The hand X-ray showed bone erosions in bilateral wrist joints. Skin biopsy revealed leukocytoclastic vasculitis. Based on these findings, he was diagnosed as malignant RA. He was successfully treated with methylprednisolone pulse therapy, cyclophosphamide and prostaglandin E 1. The second patient was 77-year old male with pneumoconiosis who experienced muscular weakness of the four limbs in proximity 4 years ago. He was diagnosed as PM based on his clinical and laboratory findings and was treated with temporary corticosteroid. He started to have polyarthralgia last year, and he was admitted because of increasing arthralgia after the treatment of pulmonary tuberculosis. The level of serum CK was slightly elevated due to hypothyroidism, and CRP was highly elevated. Rheumatoid factor and cryoglobulin were positive, but anti-Jo 1 antibody was negative. The hand X-ray showed bone erosions in bilateral wrist joints. Crystals of pyrophosphate calcium was observed in knee joints. He was diagnosed as RA associate with pseudogout. His symptoms were relieved with corticosteroid, salazosulfapyridine and anti-tuberculous therapy. These two cases had altered their clinical features from PM to definite RA, and both had pulmonary complications. Previous reports described the cases of RA followed by PM, most of which were induced by such drugs as D-penicillamine, but the cases of PM who later had developed RA are extremely unusual. The overlapped cases of RA and PM tend to highly associate with pulmonary lesions.