Structure-Guided Discovery of a Potent Inhibitor of the Ferric Citrate Binding Protein FecB in Vibrio Bacteria.

Infections caused by Gram-negative bacteria present a significant risk to human health worldwide. Novel strategies are needed to deal with the challenge caused by drug-resistant bacteria. Here, we report a new approach to combat infections by targeting iron-binding proteins to suppress bacterial growth. We investigated the function of the conserved periplasmic binding protein FecB from Vibrio alginolyticus. FecB was known to play a crucial role in the bacterial growth and to relate with biofilm formation. We then solved the crystal structures and elucidated the binding mechanism of FecB with ferric ion chelated by citrate. The results indicated that FecB binds weakly to one citrate molecule and strongly to the Fe3+-(citrate)2 complex. Based on these results, a structure-based virtual screening approach was conducted against FecB to identify small molecules that block ferric-citrate uptake. Further evaluations in vivo and in vitro demonstrated that salvianolic acid C significantly suppressed bacterial growth, indicating that targeting bacterial nutrient absorption is a promising strategy for identifying potential antibacterial drugs.

Predictive factors of nivolumab plus ipilimumab treatment efficacy in metastatic renal cell carcinoma patients.

OBJECTIVE: Nivolumab plus ipilimumab is a recommended first-line therapy regimen for metastatic renal cell carcinoma. However, it is not clear which patient characteristics are associated with its effectiveness. METHODS: We retrospectively examined 67 metastatic renal cell carcinoma patients treated with nivolumab plus ipilimumab as a first-line therapy in multiple institutions from September 2018 to August 2022. We analyzed the relationships between survival outcomes and patient-related variables, including paraneoplastic symptoms. We also analyzed the relationships between changes in symptoms and parameters and outcomes. RESULTS: Of the 67 patients, 32 patients had paraneoplastic symptoms. The median progression-free survival was 14.9 months and median overall survival was 43.3 months. The objective response rate was 49.25% (33 patients), including two patients with complete response. Patients with cytoreductive nephrectomy, bone metastasis, high C-reactive protein levels and paraneoplastic symptoms were significantly correlated with short progression-free survival in the univariate analysis. Multivariate analysis of these factors showed that the presence of paraneoplastic symptoms at treatment initiation remained an independent predictor of progression-free survival. Of the 32 patients with paraneoplastic symptoms at treatment initiation, 12 patients had symptomatic improvement and 20 did not. The 1-year progression-free survival rates were significantly longer in improved patients compared with those with no improvement. CONCLUSIONS: Patients without cytoreductive nephrectomy and with bone metastasis, liver metastasis, high C-reactive protein levels and paraneoplastic symptoms were significantly correlated with shorter progression-free survival. The presence of paraneoplastic symptoms was an independent predictor of progression-free survival. Improvement in paraneoplastic symptoms may reflect the treatment efficacy of nivolumab plus ipilimumab.

Primary signet-ring cell carcinoma of the bladder treated with laparoscopic radical cystectomy: a case report.

Primary bladder adenocarcinomas comprise 0.5-2% of all epithelial bladder neoplasms. Of these, primary signet-ring cell carcinoma of the bladder is particularly rare, accounting for 0.24% of all bladder malignancies. This tumor is frequently diagnosed at an advanced stage and has a poor prognosis. No standard treatment has yet been established. We here report a patient in whom laparoscopic cystectomy following neoadjuvant chemotherapy was effective. Our patient was a 69-year-old man who had had microscopic hematuria, undergone transurethral resection of a mass in the bladder, and been diagnosed pathologically with a primary signet-ring cell carcinoma of the bladder. No metastases were detected on computed tomography. The patient was treated with a combination of paclitaxel, cisplatin, and gemcitabine prior to undergoing laparoscopic cystectomy. The histopathological diagnosis on this operative specimen was dysplasia and no metastases were detected in the dissected lymph nodes. Complete remission has now been maintained for 9 years.

Sarcomatoid renal cell carcinoma with an inferior vena cava tumor thrombus that was completely resected by robot-assisted laparoscopic radical nephrectomy after neoadjuvant therapy nivolumab plus ipilimumab: a case report.

We here present a patient with a sarcomatoid renal cell carcinoma complicated by inferior vena cava tumor thrombus that we treated with nivolumab plus ipilimumab. This resulted in shrinkage of the tumor, enabling complete resection by robot-assisted laparoscopic radical nephrectomy. The patient is still alive with no evidence of recurrence.

Amino Acid Residues of the Metal Transporter OsNRAMP5 Responsible for Cadmium Absorption in Rice.

The transport of metals such as iron (Fe), manganese (Mn), and cadmium (Cd) in rice is highly related. Although Fe and Mn are essential elements for plant growth, Cd is a toxic element for both plants and humans. OsNRAMP5-a member of the same family as the Fe, Mn, and Cd transporter OsNRAMP1-is responsible for the transport of Mn and Cd from soil in rice. Knockout of OsNRAMP5 markedly reduces both Cd and Mn absorption, and this OsNRAMP5 knockout is indispensable for the development of low-Cd rice. However, in low-Mn environments, such plants would exhibit Mn deficiency and suppressed growth. We generated random mutations in OsNRAMP5 via error-prone PCR, and used yeast to screen for the retention of Mn absorption and the inhibition of Cd absorption. The results showed that alanine 512th is the most important amino acid residue for Cd absorption and that its substitution resulted in the absorption of Mn but not Cd.

[Clinical Outcome of Surgical Resection for Renal Cell Carcinoma with Inferior Vena Cava Tumor Thrombus].

We analyzed 45 patients who were diagnosed with renal cell carcinoma with inferior vena cava tumor thrombus (IVC) and underwent surgical resection at Nagasaki University Hospital during the 17 years from March 2003 to November 2020. The median overall survival (OS) was 68.5, 53.5, 45.7, and 20.4 months, respectively, according to the tumor thrombus level (Lv) of I, II, III and IV, with a median level of (P＝0.025). In multivariate analysis, pathological sarcomatoid changes were associated with risk of tumor recurrence in the postoperative complete remission group, and IVC thrombus level above Lv III was associated with poor prognosis in the postoperative incomplete remission group. On postoperative systemic treatment for the postoperative recurrence group and the incomplete remission group, overall survival was significantly prolonged in cases using immune checkpoint inhibitors. The results of surgical treatment of renal cell carcinoma with IVC tumor embolization were analyzed. Patients who underwent surgical resection and achieved postoperative complete remission had a relatively long prognosis with a median OS of more than 6 years. In contrast, patients with metastases, especially those with postoperative incomplete remission group, had a poor prognosis despite surgical resection, depending on the patient's situation.

Evans syndrome during pembrolizumab therapy for upper urinary tract cancer.

Introduction: Immune checkpoint inhibitors are available for the treatment of advanced urothelial carcinoma; however, serious adverse events occasionally occur. Here, we report a rare case of Evans syndrome attributed to the use of an immune checkpoint inhibitor. Case presentation: A 56-year-old man was diagnosed with left renal pelvic cancer and underwent left laparoscopic radical nephroureterectomy. Eight months postoperatively, computed tomography revealed para-aortic lymph node metastasis. Despite receiving chemotherapy, the disease progressed, and pembrolizumab was initiated. After 26 months of pembrolizumab treatment, the patient developed fever and anemia. Hematologic examination confirmed the diagnosis of Evans syndrome. He was treated with blood transfusions and corticosteroids, and gradual symptom improvement was observed. Conclusion: This report highlights the potential risk of Evans syndrome associated with immune checkpoint inhibitor treatment. Clinicians should be aware of this possibility and consider early intervention with corticosteroids.

Concomitant Activation of OsNAS2 and OsNAS3 Contributes to the Enhanced Accumulation of Iron and Zinc in Rice.

Nicotianamine (NA) is produced by NA synthase (NAS), which contains three genes in rice and is responsible for chelating metals such as iron (Fe) and zinc (Zn), as well as preserving metal homeostasis. In this study, we generated a transgenic plant (23D) that shows simultaneous activation of OsNAS2 and OsNAS3 by crossing two previously identified activation-tagged mutants, OsNAS2-D1 (2D) and OsNAS3-D1 (3D). Concomitant activation of both genes resulted in the highest Fe and Zn concentrations in shoots and roots of the 23D plants grown under normal conditions and Fe and Zn limited growth conditions. Expression of genes for the biosynthesis of mugineic acid family phytosiderophores (MAs) and Fe and Zn uptake were enhanced in 23D roots. Additionally, 23D plants displayed superior growth to other plants at higher pH levels. Importantly, 23D seeds had NA and 2'-deoxymugineic acid (DMA) concentrations that were 50.6- and 10.0-fold higher than those of the WT. As a result, the mature grain Fe and Zn concentrations of the 23D plant were 4.0 and 3.5 times greater, respectively, than those of the WT. Furthermore, 23D plants exhibited the greatest resistance to excess metals. Our research suggests that simultaneous activation of OsNAS2 and OsNAS3 can enhance Fe and Zn accumulation in rice grains while also increasing plant tolerance to growing situations with metal deficiency and excess metal availability.

[A Retrospective Study on the Malignancies after Renal Transplantation].

Detection of post-transplant malignant tumors and the analysis of the associated risk factors is important for monitoring the progress after renal transplantation. In this study, we retrospectively examined the medical records of 298 patients who underwent renal transplantation at two facilities in Nagasaki Prefecture (Nagasaki University Hospital and National Hospital Organization Nagasaki Medical Center). Of the 298 patients, 45 (15.1%) patients had developed malignant tumors with 50 lesions. The most common type of malignant tumor was skin cancer (eight patients; 17.8%), followed by renal cancer (six patients; 13.3%), and pancreatic cancer and colorectal cancer, (four patients; 9.0% each). Five patients (11.1%) had multiple cancers, four of whom had skin cancer. The cumulative incidence within 10 and 20 years after renal transplantation was 6.0 and 17.9%, respectively. Univariate analysis identified age at transplantation and administration of cyclosporine and rituximab as risk factors, while multivariate analysis identified age at transplantation and administration of rituximab as independent factors. The administration of rituximab was associated with the development of malignant tumors. However, further investigation is required to establish the association with post-transplant malignant neoplasms.

Initial experience in robot-associated radical nephrectomy with inferior vena cava tumor thrombectomy.

Background: Renal cell carcinoma (RCC) is often locally invasive and may extend from the renal vein into the inferior vena cava (IVC) as a venous tumor thrombus (VTT). Radical nephrectomy with IVC tumor thrombectomy (IVC-TT) via an open approach has been shown to carry high morbidity and mortality. Recently, robot-associated radical nephrectomy (RARN) has been developed with the aim of improving the performance and outcomes of surgery for RCC with IVC-VTT. Methods: We here present four patients who had right RCC with IVC-VTT and underwent RARN with IVC-TT in Nagasaki University Hospital. All four patients had level II IVC-TT and underwent RARN with IVC-TT using a da Vinci Xi surgical system. The procedure comprised performing the Kocher maneuver, exposing the right renal artery in the aortocaval region dorsal to the left renal vein, exposing, mobilizing, and clipping the IVC, clamping and incising the IVC, and removing the kidney with the VTT en bloc. Results: The mean tumor size was 83.1 (range, 50.1-115.2) mm and the mean length of the VTT within the IVC 41.6 (range, 25.3-44.3) mm. The mean console time was 290 (range, 287-367) minutes and the mean blood loss was 200 (range, 175-260) mL and no patient required blood transfusion. Conclusions: Our initial experience of the procedure of RARN with IVC-TT for level II IVC-VTT is that it is safe and has acceptable perioperative outcomes and complications.

Preservation of Split Renal Function After Laparoscopic and Robot-assisted Partial Nephrectomy.

BACKGROUND/AIM: To analyze the effects of laparoscopic partial nephrectomy (LPN) and robot-assisted partial nephrectomy (RAPN) for the treatment of renal cell carcinoma (RCC) on subsequent split renal function using renal scintigraphy. PATIENTS AND METHODS: We retrospectively analyzed data from 174 patients who underwent LPN or RAPN by a single surgeon, and assessed their total and split renal function before and 6 months after each procedure. Split renal function was analyzed using 99mTc-2,3 dimercaptosuccinic acid renal imaging and calculated as the total estimated glomerular filtration rate (eGFR) × uptake ratio on the surgical side/uptake ratio on the contralateral side. RESULTS: LPN or RAPN were performed in 51 (29.3%) and 123 (70.7%) participants, respectively. Their median eGFRs before and after surgery were 32.76 and 27.74 ml/min/1.73 m2, respectively, and 70 of them (40.2%) showed a preservation of split eGFR of >90%, which was used to define a successful procedure. Participants who underwent a successful procedure had significantly lower RENAL nephrometry scoring system (RNS) scores and fewer of them had external tumors. Successful procedures were associated with shorter warm ischemia time, were more likely to be RAPN, and less likely to involve parenchymal suturing. Multivariate analysis showed that a low RNS score and parenchymal suturing were significant independent predictors of split renal function following partial nephrectomy (PN). CONCLUSION: Preoperative RNS score and the use of parenchymal suturing are significantly associated with a preservation of split renal function of >90% in patients who undergo PN for the treatment of RCC.

[Renal Cell Carcinomas in Patients on Chronic Dialysis].

Patients on chronic dialysis for end-stage renal disease (ESRD) show an increased incidence of renal cell carcinoma (RCC). We investigated the clinicopathological characteristics and outcomes of 54 patients who underwent nephrectomy for RCC due to ESRD between 1992 and 2019. The patients consisted of 44 men and 10 women, with a median age of 62.9 years. The median duration of dialysis before surgery was 12.9 years. The clinical stage of the 54 RCCs was stage I in 44, stage II in 1, stage III in 1, and stage IV in 8. With a median follow-up of 5.1 years after surgery, the 5-year cancer-specific and overall survival rates were 84.3 and 61.8%, respectively. Patients with symptomatic RCC had a longer period of dialysis, presented with larger tumors of higher grade and stage, and had worse prognosis compared with those with incidentally discovered RCC. Cox proportional hazards analysis performed with clinicopathological features and symptomatic/incidental detection showed that older age and symptomatic RCC were independently associated with worse overall survival. Our data show that early detection is important for a good prognosis.

Development of a mugineic acid family phytosiderophore analog as an iron fertilizer.

Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)•2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.

Defects in the rice aconitase-encoding OsACO1 gene alter iron homeostasis.

KEY MESSAGE: Rice aconitase gene OsACO1 is involved in the iron deficiency-signaling pathway for the expression of iron deficiency-inducible genes, either thorough enzyme activity or possible specific RNA binding for post-transcriptional regulation. Iron (Fe) is an essential element for virtually all living organisms. When plants are deficient in Fe, Fe acquisition systems are activated to maintain Fe homeostasis, and this regulation is mainly executed at the gene transcription level. Many molecules responsible for Fe uptake, translocation, and storage in plants have been identified and characterized. However, how plants sense Fe status within cells and then induce a transcriptional response is still unclear. In the present study, we found that knockdown of the OsACO1 gene, which encodes an aconitase in rice, leads to the down-regulation of selected Fe deficiency-inducible genes involved in Fe uptake and translocation in roots, and a decrease in Fe concentration in leaves, even when grown under Fe-sufficient conditions. OsACO1 knockdown plants showed a delayed transcriptional response to Fe deficiency compared to wild-type plants. In contrast, overexpression of OsACO1 resulted in the opposite effects. These results suggest that OsACO1 is situated upstream of the Fe deficiency-signaling pathway. Furthermore, we found that the OsACO1 protein potentially has RNA-binding activity. In vitro screening of RNA interactions with OsACO1 revealed that RNA potentially forms a unique stem-loop structure that interacts with OsACO1 via a conserved GGUGG motif within the loop structure. These results suggest that OsACO1 regulate Fe deficiency response either thorough enzyme activity catalyzing isomerization of citrate, or specific RNA binding for post-transcriptional regulation.

Elevated Expression of Vacuolar Nickel Transporter Gene IREG2 Is Associated With Reduced Root-to-Shoot Nickel Translocation in Noccaea japonica.

A number of metal hyperaccumulator plants, including nickel (Ni) hyperaccumulators, have been identified in the genus Noccaea. The ability to accumulate Ni in shoots varies widely among species and ecotypes in this genus; however, little is known about the molecular mechanisms underlying this intra- and inter-specific variation. Here, in hydroponic culture, we compared Ni accumulation patterns between Noccaea japonica, which originated in Ni-enriched serpentine soils in Mt. Yubari (Hokkaido, Japan), and Noccaea caerulescens ecotype Ganges, which originated in zinc/lead-mine soils in Southern France. Both Noccaea species showed extremely high Ni tolerance compared with that of the non-accumulator Arabidopsis thaliana. But, following treatment with 200 µM Ni, N. caerulescens showed leaf chlorosis, whereas N. japonica did not show any stress symptoms. Shoot Ni concentration was higher in N. caerulescens than in N. japonica; this difference was due to higher efficiency of root-to-shoot Ni translocation in N. caerulescens than N. japonica. It is known that the vacuole Ni transporter IREG2 suppresses Ni translocation from roots to shoots by sequestering Ni in the root vacuoles. The expression level of the IREG2 gene in the roots of N. japonica was 10-fold that in the roots of N. caerulescens. Moreover, the copy number of IREG2 per genome was higher in N. japonica than in N. caerulescens, suggesting that IREG2 expression is elevated by gene multiplication in N. japonica. The heterologous expression of IREG2 of N. japonica and N. caerulescens in yeast and A. thaliana confirmed that both IREG2 genes encode functional vacuole Ni transporters. Taking these results together, we hypothesize that the elevation of IREG2 expression by gene multiplication causes the lower root-to-shoot Ni translocation in N. japonica.

Chemical Speciation, Plant Uptake, and Toxicity of Heavy Metals in Agricultural Soils.

Heavy metals in agricultural soils exist in diverse dissolved (free cations and complexed species of positive, neutral, or negative charges), particulate (sorbed, structural, and coprecipitated), and colloidal (micro- and nanometer-sized particles) species. The fate of different heavy metal species is controlled by the master variables: pH (solubility), ionic strength (activity and charge-shielding), and dissolved organic carbon (complexation). In the rhizosphere, chemical speciation controls toxicokinetics (uptake and transport of metals by plants) while toxicodynamics (interaction between the plant and absorbed species) drives the toxicity outcome. Based on the critical review, the authors recommend omics and data mining techniques to link discrete knowledge bases from the speciation dynamics, soil microbiome, and plant transporter/gene expression relevant to homeostasis conditions of modern agriculture. Such efforts could offer a disruptive application tool to improve and sustain plant tolerance, food safety, and environmental quality.

Characterization of the Nicotianamine Exporter ENA1 in Rice.

Under iron (Fe) deficiency, graminaceous plants produce and secrete Fe-chelating phytosiderophores of the mugineic acid (MA) family into the rhizosphere to solubilize and mediate uptake of sparingly soluble Fe in the soil. MAs and their biosynthetic intermediate, nicotianamine (NA), are also important for the translocation of divalent metals such as Fe and zinc (Zn) throughout the plant body. In this study, the physiological role of the efflux transporter EFFLUX TRANSPORTER OF NA (ENA1), which exports NA out of cells, was analyzed in rice. Promoter-GUS analysis showed that ENA1 was mainly expressed in roots, and strongly upregulated under Fe-deficient conditions. In epidermal onion cells and rice roots, green fluorescent protein-tagged ENA1 localized mainly to the plasma membrane, while a part of the fluorescence was observed in vesicular structures in the cytoplasm. In the younger stage after germination, ENA1-overexpressing rice plants exhibited truncated roots with many root hairs compared to wild-type plants, while these phenotype were not observed in high Zn-containing medium. In Arabidopsis, which use a different strategy for Fe uptake from rice, ENA1 overexpression did not show any apparent phenotypes. Oligo DNA microarray analysis in rice showed that ENA1 knockout affects the response to stress, especially in root plastids. These results suggest that ENA1 might be recycling between the plasma membrane and cellular compartments by vesicular transport, playing an important role in the transport of NA, which is important for the physiological response to Fe deficiency.

Prediction of Nonadherence and Renal Prognosis by Pre-Transplantation Serum Phosphate Levels.

BACKGROUND Identifying characteristics of patients at high risk of poor adherence before transplantation would be advantageous. However, the optimal approach for characterizing such patients remains unknown. We aimed to evaluate the association between factors for hemodialysis nonadherence and post-transplant renal prognosis. We hypothesized that these factors would influence post-transplantation adherence and worsen renal prognosis. MATERIAL AND METHODS We reviewed patients on hemodialysis who underwent kidney transplantation at our hospital between 2000 and 2017 to identify risk factors associated with poor prognosis. The patients' background and pre-transplantation data, known hemodialysis nonadherence factors, serum phosphate and potassium levels, and interdialytic weight gains were evaluated. The primary endpoint was renal death. We also evaluated the fluctuation of calcineurin inhibitor concentration and weight gain after transplantation. RESULTS Seventy-seven patients were eligible, and the mean observational period was 83.2 months (standard deviation, 50.5). Thirteen patients reached the endpoint. Cox proportional hazards regression analysis showed that pre-transplantation serum phosphate level was a risk factor for renal death (p<0.05), while serum potassium levels and weight gain were not. In addition, fluctuation of calcineurin inhibitor concentration was observed in patients with higher phosphate levels before transplantation (p=0.03). Weight gain after transplantation was not associated with the hemodialysis nonadherence factors. CONCLUSIONS High pre-transplantation serum phosphate levels are considered to represent poor drug adherence and/or an unhealthy lifestyle. Patient education that conveys the importance of adhering to medications and provides nutritional guidance is crucial for improving post-transplantation renal prognosis.

From Leguminosae/Gramineae Intercropping Systems to See Benefits of Intercropping on Iron Nutrition.

To achieve sustainable development with a growing population while sustaining natural resources, a sustainable intensification of agriculture is necessary. Intercropping is useful for low-input/resource-limited agricultural systems. Iron (Fe) deficiency is a worldwide agricultural problem owing to the low solubility and bioavailability of Fe in alkaline and calcareous soils. Here, we summarize the effects of intercropping systems on Fe nutrition. Several cases showed that intercropping with graminaceous plants could be used to correct Fe nutrition of Leguminosae such as peanut and soybean or fruits such as Psidium guajava L., Citrus, grape and pear in calcareous soils. Intercropping systems have strong positive effects on the physicochemical and biochemical characteristics of soil and the microbial community due to interspecific differences and interactions in the rhizosphere. Rhizosphere interactions can increase the bioavailability of Fe with the help of phytosiderophores. Enriched microorganisms may also facilitate the Fe nutrition of crops. A peanut/maize intercropping system could help us understand the dynamics in rhizosphere and molecular mechanism. However, the role of microbiome in regulating Fe acquisition of root and the mechanisms underlying these phenomena in other intercropping system except peanut/maize need further work, which will help better utilize intercropping to increase the efficiency of Fe foraging.

The Yellow Stripe-Like (YSL) Gene Functions in Internal Copper Transport in Peanut.

Copper (Cu) is involved in fundamental biological processes for plant growth and development. However, Cu excess is harmful to plants. Thus, Cu in plant tissues must be tightly regulated. In this study, we found that the peanut Yellow Stripe-Like family gene AhYSL3.1 is involved in Cu transport. Among five AhYSL genes, AhYSL3.1 and AhYSL3.2 were upregulated by Cu deficiency in peanut roots and expressed mainly in young leaves. A yeast complementation assay suggested that the plasma membrane-localized AhYSL3.1 was a Cu-nicotianamine complex transporter. High expression of AhYSL3.1 in tobacco and rice plants with excess Cu resulted in a low concentration of Cu in young leaves. These transgenic plants were resistant to excess Cu. The above results suggest that AhYSL3.1 is responsible for the internal transport of Cu in peanut.