When pancreata fly: Outcomes and lessons learned from the development of a Pancreas Transplant Import Program.

BACKGROUND: To address long waitlist times and increase pancreas transplantation, our center has implemented a protocol for long-distance importation of pancreata. METHODS: We conducted a retrospective review of pancreas transplantation at our institution from January 1, 2014, the start of our importation program, through September 30, 2021. Outcomes were compared between locally procured grafts and imported grafts, defined as grafts procured greater than 250 nautical miles (NM) from our center. RESULTS: Eighty-one patients underwent pancreas transplantation during the study time period; 19 (23.5%) received imported grafts. There were no significant differences in recipient demographics or type of transplant received. Mean distance of import was 644.2 ± 234.0 NM. Imported grafts were more likely to be from pediatric donors <18 years old (p = .02) and a significantly higher proportion of imported grafts came from donors weighing <30 kg (26.3 vs. 3.2%, p = .007). Cold ischemic time was longer for imported grafts than for local grafts (13.4 ± 2.3 h vs. 9.8 ± 2.2 h, p < .01). There was no significant difference in deaths or graft losses within 90 days or at 1 year between groups. CONCLUSION: Centers should consider expanding criteria for acceptance of imported pancreata to increase the number of transplants and combat organ nonutilization.

Metronidazole for the treatment of cutaneous vulval Crohn disease: A systematic review.

Background: Cutaneous vulval Crohn disease (VCD) is an under-recognised extra-intestinal manifestation of Crohn disease (CD) which is challenging to identify and treat. It causes significant oedema, painful deep fissures, and has potential to cause permanent disfiguring changes to vulval anatomy. There is no agreement on the best management for VCD. Objectives: This systematic review evaluates the use of metronidazole for the treatment of VCD in women and children. Methods: We conducted a systematic review (PROSPERO CRD42021285033) of the use of metronidazole in clinically or histologically diagnosed non-contiguous VCD in patients of all ages and ethnicities. We recorded clinical improvement, reduction in flares, relapse and adverse events using a standardised form. Results: 49 records (40 case reports and 9 case series) met inclusion criteria, comprising a total of 57 patients with an age range of 5-61 years. The most reported presenting features in VCD were: oedema, erythema, ulcers/fissures and induration/thickening. Gastrointestinal CD was present in 33/57 (58%). Vulval biopsies were undertaken in 47/57 (83%). Daily doses ranged from 250 to 1500 mg with treatment duration 8 days to 18 months. Improvement of any magnitude was observed in 40/57 (70%) cases. Relapse was described in 11/57 (19%) cases. No response/worsening was reported in 17/57 (30%) cases. Adverse events occurred in two patients. Conclusion: Metronidazole appears to be useful in managing VCD, either as a primary treatment or adjunctive therapy. However, the evidence is insufficient for firm conclusions to be drawn. Further studies including randomised controlled trials are recommended.

The best insulin delivery is a human pancreas.

PURPOSE: We wanted to compare glycemic control post pancreas transplantation with newer therapeutic options. METHODS: We conducted a retrospective analysis of pancreas transplantation at our institution from January 1, 2008, through September 30, 2021. All patients who underwent pancreatic transplantation were 18 years and older. We compared pre-transplant glycemic control of those patients, whether self-monitoring or continuous glucose monitor to their post-transplant glycemic control. Outcomes were assessed by HgbA1C level at evaluation (eval), pretransplant (pre), within the first 5 months posttransplant (post) and 1 year post transplant (1 year). RESULTS: One hundred and thirty-four patients underwent pancreas transplantation during the 14-year study period. Overall, 1-year patient and graft survival were 95% and 88%. The mean HgbA1C (%) for eval and pre were 8.5(SD ± 1.7) and 8.3(SD ± 1.7), which was significantly higher than post, and 1 year at 5.1(SD ± .6, p < .01) and 5.2(SD ± .6, p < .01). Of those, 38 patients presented with continuous glucose monitors (CGM) +/- pump. Their mean HgbA1C(%) was 8.2(SD ± 1.5) at eval 8.1(SD ± 1.3). These were also significantly higher than post 5.0(SD ± .6, p < .01), and 1 year 5.1(SD ± .5, p < .01). CONCLUSION: Pancreas transplant provides superior glycemic control to continuous glucose monitoring and remains the optimal therapy for appropriately selected patients with diabetes.

Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

Access to kidney transplantation among pediatric candidates with prior solid organ transplants in the United States.

BACKGROUND: Pediatric kidney transplant candidates require timely access to transplant to optimize growth and neurodevelopmental outcomes. We studied access to transplant for pediatric candidates with prior organ transplants. METHODS: We used US registry data to identify pediatric kidney transplant candidates added to the waiting list 2015-2019 and used competing risk regression to study the association between prior transplant status and probability of receiving a kidney transplant, treating wait-list removal and death as competing events. RESULTS: Of 4962 pediatric kidney transplant candidates included, 89% had no prior transplant and 11% had received a prior organ transplant (kidney 87%, liver 5%, heart 5%). Prior transplant recipients were older at listing (median 15 vs. 12 years) and more likely to have PRA≥98% (22% vs. 0.3%) (both p < .001). There was no significant difference in the proportion of candidates from each group who were preemptively wait-listed. Unadjusted competing risk regression showed a lower risk of kidney transplant after wait-listing among candidates with prior organ transplant (HR 0.52, 95%CI 0.47-0.59, p < .001). This association remained significant after adjusting for candidate characteristics (HR 0.73, 95%CI 0.63-0.83, p < .001). Among deceased donor kidney recipients, median KDPI was similar between groups, but recipients with prior transplants were more likely to receive kidneys from donors with hypertension (4% vs. 1%, p = .01) and donors after cardiac death (11% vs. 4%, p < .001). CONCLUSIONS: Pediatric kidney transplant candidates with prior organ transplants have reduced access to transplant after wait-listing. Allocation system changes are needed to improve timely access to transplant for this vulnerable group.

PfMDR1 Transport Rates Assessed in Intact Isolated Plasmodium falciparum Digestive Vacuoles Reflect Functional Drug Resistance Relationship with pfmdr1 Mutations.

Drug resistance often emerges from mutations in solute transporters. Single amino acid exchanges may alter functionality of transporters with 'de novo' ability to transport drugs away from their site of action. The PfMDR1 transporter (or P-glycoprotein 1) is located in the membrane of the digestive vacuole (DV), functions as an ATP-dependent pump, and transports substrates into the DV. In this study, four strains of Plasmodium falciparum, carrying various pfmdr1 gene mutations, were analysed for their transport characteristics of Fluo-4 in isolated DVs of parasites. To obtain quantitative estimates for PfMDR1 DV surface expression, PfMDR1 protein amounts on each strain's DV membrane were evaluated by quantitative ELISA. Fluo-4, acting as a substrate for PfMDR1, was applied in DV uptake assays ('reverse Ca2+ imaging'). Viable DVs were isolated from trophozoite stages with preserved PfMDR1 activity. This newly developed assay enabled us to measure the number of Fluo-4 molecules actively transported into isolated DVs per PfMDR1 molecule. The drug-resistant strain Dd2 presented the highest transport rates, followed by K1 and the drug-sensitive strain 3D7, compatible with their copy numbers. With this assay, an evaluation of the probability of resistance formation for newly developed drugs can be implemented in early stages of drug development.

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search.

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.

Improved stability of polyclonal antibodies: A case study with lyophilization-conserved antibodies raised against epitopes from the malaria parasite Plasmodium falciparum.

Antibodies can be produced as polyclonal (pAb) or monoclonal (mAb) liquid formulations with limited shelf-life. For pAbs, unlike mAbs, only little is known about excipients and lyophilization affecting antibody stability upon reconstitution. We used a model pAb directed against Plasmodium falciparum (Pf) pyridoxal 5'-phosphate synthase 2 (Pdx2) to systemically study effects of bulking agents (amino acids, phosphate buffers, salt solutions), sugar(alcohols), surfactants and protein additions (bovine serum albumin, BSA) in liquid pAb formulations (isolated or in combinations) on the activity to detect the antigen in Pf extracts by Western blots. Repeated freeze-thaw cycles (20x) and extended room temperature storage markedly compromised pAb stability, the former being ameliorated by addition of cryoprotectants (glycerol, sucrose). Lyophilization of pure liquid pAb formulation markedly decreased antibody reactivity upon reconstitution which was not preserved by most bulking agents tested (e.g., histidine, arginine, acetate). Among the tested salt solutions (NaCl, Ringer, PBS), phosphate buffered saline had the largest lyoprotective potential, alongside sucrose, but not trehalose or maltitol. Among combinations of excipients, PBS, sucrose, low concentration BSA and Tween potently preserved PfPdx2 stability. Results for PBS were transferable to PfEnolase pAb, indicating that some of the formulations investigated here might be a low-cost solution for more general applicability to pAbs.

Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria.

A substantial challenge worldwide is emergent drug resistance in malaria parasites against approved drugs, such as chloroquine (CQ). To address these unsolved CQ resistance issues, only rare examples of artemisinin (ART)-based hybrids have been reported. Moreover, protein targets of such hybrids have not been identified yet, and the reason for the superior efficacy of these hybrids is still not known. Herein, we report the synthesis of novel ART-isoquinoline and ART-quinoline hybrids showing highly improved potencies against CQ-resistant and multidrug-resistant P. falciparum strains (EC50 (Dd2) down to 1.0 nm; EC50 (K1) down to 0.78 nm) compared to CQ (EC50 (Dd2)=165.3 nm; EC50 (K1)=302.8 nm) and strongly suppressing parasitemia in experimental malaria. These new compounds are easily accessible by step-economic C-H activation and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reactions. Through chemical proteomics, putatively hybrid-binding protein targets of the ART-quinolines were successfully identified in addition to known targets of quinoline and artemisinin alone, suggesting that the hybrids act through multiple modes of action to overcome resistance.

Spontaneous formation of fluid escape pipes from subsurface reservoirs.

Ubiquitous observations of channelised fluid flow in the form of pipes or chimney-like features in sedimentary sequences provide strong evidence for significant transient permeability-generation in the subsurface. Understanding the mechanisms and dynamics for spontaneous flow localisation into fluid conductive chimneys is vital for natural fluid migration and anthropogenic fluid and gas operations, and in waste sequestration. Yet no model exists that can predict how, when, or where these conduits form. Here we propose a physical mechanism and show that pipes and chimneys can form spontaneously through hydro-mechanical coupling between fluid flow and solid deformation. By resolving both fluid flow and shear deformation of the matrix in three dimensions, we predict fluid flux and matrix stress distribution over time. The pipes constitute efficient fluid pathways with permeability enhancement exceeding three orders of magnitude. We find that in essentially impermeable shale (10-19 m2), vertical fluid migration rates in the high-permeability pipes or chimneys approach rates expected in permeable sandstones (10-15 m2). This previously unidentified fluid focusing mechanism bridges the gap between observations and established conceptual models for overcoming and destroying assumed impermeable barriers. This mechanism therefore has a profound impact on assessing the evolution of leakage pathways in natural gas emissions, for reliable risk assessment for long-term subsurface waste storage, or CO2 sequestration.

Quantification of human complement factor H binding to asexual malaria blood stages by an enzyme-linked immunosorbent assay.

The human complement system is the most effective defense mechanism of the human innate immune system. One major negative regulator of the alternative pathway in human blood is complement factor H (FH). It binds to autologous cells and thus, prevents complement attack against body-cells or tissues. Various pathogens are known to escape complement recognition by recruiting FH to provide protection against the host's immune system. This immune evasion mechanism was recently qualitatively reported for asexual malaria blood stages. To indirectly evaluate the stage-specific potential of FH-receptor proteins as vaccine candidates, we quantified the FH molecules bound to the surface of different malaria blood stage parasites by Western blot and a commercially available FH-ELISA, which was originally designed to measure the FH concentration in human serum. Host-cell-free merozoites and intracellular mature schizont (here called segmenter) stages bind significantly more FH molecules than earlier parasite stages.

SYBR® Green I-Based Fluorescence Assay to Assess Cell Viability of Malaria Parasites for Routine Use in Compound Screening.

Owing to its fast and reliable assessment of parasite growth, the SYBR® Green I-based fluorescence assay is widely used to monitor drug susceptibility of malaria parasites. Its particular advantages are that it is a simple, one-step procedure and very cost-effective making it especially suited for high through put screening of newly developed drugs and drug combinations. Here we describe a SYBR® Green I-based fluorescence assay protocol to be used for routine screening of compounds and extracts in a research laboratory environment. A variation of the standard protocol is also provided allowing to address stage-specific effects of fast-acting drugs.

Structural Insights into the Recognition of N2 -Aryl- and C8-Aryl DNA Lesions by the Repair Protein XPA/Rad14.

Aromatic amines are strongly carcinogenic. They are activated in the liver to give reactive nitrenium ions that react with nucleobases within the DNA duplex. The reaction occurs predominantly at the C8 position of the dG base, thereby giving C8-acetyl-aryl- or C8-aryl-dG adducts in an electrophilic aromatic substitution reaction. Alternatively, reaction with the exocyclic 2-NH2 group is observed. Although the C8 adducts retain base-pairing properties, base pairing is strongly compromised in the case of the N2 adducts. Here we show crystal structures of two DNA lesions, N2 -acetylnaphthyl-dG and C8-fluorenyl-dG, within a DNA duplex recognized by the repair protein Rad14. The structures confirm that two molecules of the repair protein recognize the lesion and induce a 72 or 78° kink at the site of the damage. Importantly, the same overall kinked structure is induced by binding of the repair proteins, although the structurally different lesions result in distinct stacking interactions of the lesions within the duplex. The results suggest that the repair protein XPA/Rad14 is a sensor that recognizes flexibility. The protein converts the information that structurally different lesions are present in the duplex into a unifying sharply kinked recognition motif.

Molecular mechanisms of xeroderma pigmentosum (XP) proteins.

Nucleotide excision repair (NER) is a highly versatile and efficient DNA repair process, which is responsible for the removal of a large number of structurally diverse DNA lesions. Its extreme broad substrate specificity ranges from DNA damages formed upon exposure to ultraviolet radiation to numerous bulky DNA adducts induced by mutagenic environmental chemicals and cytotoxic drugs used in chemotherapy. Defective NER leads to serious diseases, such as xeroderma pigmentosum (XP). Eight XP complementation groups are known of which seven (XPA-XPG) are caused by mutations in genes involved in the NER process. The eighth gene, XPV, codes for the DNA polymerase ɳ, which replicates through DNA lesions in a process called translesion synthesis (TLS). Over the past decade, detailed structural information of these DNA repair proteins involved in eukaryotic NER and TLS have emerged. These structures allow us now to understand the molecular mechanism of the NER and TLS processes in quite some detail and we have begun to understand the broad substrate specificity of NER. In this review, we aim to highlight recent advances in the process of damage recognition and repair as well as damage tolerance by the XP proteins.

Structural Basis for Bulky-Adduct DNA-Lesion Recognition by the Nucleotide Excision Repair Protein Rad14.

Heterocyclic aromatic amines react with purine bases and result in bulky DNA adducts that cause mutations. Such structurally diverse lesions are substrates for the nucleotide excision repair (NER). It is thought that the NER machinery recognises and verifies distorted DNA conformations, also involving the xeroderma pigmentosum group A and C proteins (XPA, XPC) that act as a scaffold between the DNA substrate and several other NER proteins. Here we present the synthesis of DNA molecules containing the polycyclic, aromatic amine C8-guanine lesions acetylaminophenyl, acetylaminonaphthyl, acetylaminoanthryl, and acetylaminopyrenyl, as well as their crystal structures in complex with the yeast XPA homologue Rad14. This work further substantiates the indirect lesion-detection mechanism employed by the NER system that recognises destabilised and deformable DNA structures.

A WD40-repeat protein unique to malaria parasites associates with adhesion protein complexes and is crucial for blood stage progeny.

BACKGROUND: During development in human erythrocytes, Plasmodium falciparum parasites display a remarkable number of adhesive proteins on their plasma membrane. In the invasive merozoites, these include members of the PfMSP1 and PfAMA1/RON complexes, which facilitate contact between merozoites and red blood cells. In gametocytes, sexual precursor cells mediating parasite transmission to the mosquito vector, plasma membrane-associated proteins primarily belong to the PfCCp and 6-cys families with roles in fertilization. This study describes a newly identified WD40-repeat protein unique to Plasmodium species that associates with adhesion protein complexes of both merozoites and gametocytes. METHODS: The WD40-repeat protein-like protein PfWLP1 was identified via co-immunoprecipitation assays followed by mass spectrometry and characterized using biochemical and immunohistochemistry methods. Reverse genetics were employed for functional analysis. RESULTS: PfWLP1 is expressed both in schizonts and gametocytes. In mature schizonts, the protein localizes underneath the merozoite micronemes and interacts with PfAMA1, while in gametocytes PfWLP1 primarily accumulates underneath the plasma membrane and associates with PfCCp1 and Pfs230. Reverse genetics failed to disrupt the pfwlp1 gene, while haemagglutinin-tagging was feasible, suggesting a crucial function for PfWLP1 during blood stage replication. CONCLUSIONS: This is the first report on a plasmodial WD40-repeat protein associating with cell adhesion proteins. Since WD40 domains are known to mediate protein-protein contact by serving as a rigid scaffold for protein interactions, the presented data suggest that PfWLP1 supports the stability of adhesion protein complexes of the plasmodial blood stages.

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA).

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a ß-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut.

Human complement is a first line defense against infection in which circulating proteins initiate an enzyme cascade on the microbial surface that leads to phagocytosis and lysis. Various pathogens evade complement recognition by binding to regulator proteins that protect host cells from complement activation. We show that emerging gametes of the malaria parasite Plasmodium falciparum bind the host complement regulator factor H (FH) following transmission to the mosquito to protect from complement-mediated lysis by the blood meal. Human complement is active in the mosquito midgut for approximately 1 hr postfeeding. During this period, the gamete surface protein PfGAP50 binds to FH and uses surface-bound FH to inactivate the complement protein C3b. Loss of FH-mediated protection, either through neutralization of FH or blockade of PfGAP50, significantly impairs gametogenesis and inhibits parasite transmission to the mosquito. Thus, Plasmodium co-opts the protective host protein FH to evade complement-mediated lysis within the mosquito midgut.

Family members stick together: multi-protein complexes of malaria parasites.

Malaria parasites express a broad repertoire of proteins whose expression is tightly regulated depending on the life-cycle stage of the parasite and the environment of target organs in the respective host. Transmission of malaria parasites from the human to the anopheline mosquito is mediated by intraerythrocytic sexual stages, termed gametocytes, which circulate in the peripheral blood and are essential for the spread of the tropical disease. In Plasmodium falciparum, gametocytes express numerous extracellular proteins with adhesive motifs, which might mediate important interactions during transmission. Among these is a family of six secreted proteins with adhesive modules, termed PfCCp proteins, which are highly conserved throughout the apicomplexan clade. In P. falciparum, the proteins are expressed in the parasitophorous vacuole of gametocytes and are subsequently exposed on the surface of macrogametes during parasite reproduction in the mosquito midgut. One characteristic of the family is a co-dependent expression, such that loss of all six proteins occurs if expression of one member is disrupted via gene knockout. The six PfCCp proteins interact by adhesion domain-mediated binding and thus form complexes on the sexual stage surface having adhesive properties. To date, the PfCCp proteins represent the only protein family of the malaria parasite sexual stages that assembles to multimeric complexes, and only a small number of such protein complexes have so far been identified in other life-cycle stages of the parasite.