Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.

Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles, biased T cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases.

Integrating Upper-Limb Prostheses with the Human Body: Technology Advances, Readiness, and Roles in Human-Prosthesis Interaction.

Significant advances in bionic prosthetics have occurred in the past two decades. The field's rapid expansion has yielded many exciting technologies that can enhance the physical, functional, and cognitive integration of a prosthetic limb with a human. We review advances in the engineering of prosthetic devices and their interfaces with the human nervous system, as well as various surgical techniques for altering human neuromusculoskeletal systems for seamless human-prosthesis integration. We discuss significant advancements in research and clinical translation, focusing on upper limbprosthetics since they heavily rely on user intent for daily operation, although many discussed technologies have been extended to lower limb prostheses as well. In addition, our review emphasizes the roles of advanced prosthetics technologies in complex interactions with humans and the technology readiness levels (TRLs) of individual research advances. Finally, we discuss current gaps and controversies in the field and point out future research directions, guided by TRLs.

High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST.

The accurate modeling of energetic contributions to protein structure is a fundamental challenge in computational approaches to protein analysis and design. We describe a general computational method, EmCAST (empirical Cα stabilization), to score and optimize the sequence to the structure in proteins. The method relies on an empirical potential derived from the database of the Cα dihedral angle preferences for all possible four-residue sequences, using the data available in the Protein Data Bank. Our method produces stability predictions that naturally correlate one-to-one with the experimental results for solvent-exposed mutation sites. EmCAST predicted four mutations that increased the stability of a three-helix bundle, UBA(1), from 2.4 to 4.8 kcal/mol by optimizing residues in both helices and turns. For a set of eight variants, the predicted and experimental stabilizations correlate very well (R2 = 0.97) with a slope near 1 and with a 0.16 kcal/mol standard error for EmCAST predictions. Tests against literature data for the stability effects of surface-exposed mutations show that EmCAST outperforms the existing stability prediction methods. UBA(1) variants were crystallized to verify and analyze their structures at an atomic resolution. Thermodynamic and kinetic folding experiments were performed to determine the magnitude and mechanism of stabilization. Our method has the potential to enable the rapid, rational optimization of natural proteins, expand the analysis of the sequence/structure relationship, and supplement the existing protein design strategies.

Lung cancer-kidney cross talk induces kidney injury, interstitial fibrosis, and enhances cisplatin-induced nephrotoxicity.

Patients with cancer represent a unique patient population with increased susceptibility to kidney disease. Drug-induced acute kidney injury (AKI) in patients with cancer is a common problem. Cisplatin is a highly effective treatment used in many solid-organ cancers and causes AKI in 30% of patients, increasing the risk of chronic kidney disease development. Most preclinical cisplatin toxicity studies have been completed in mice without cancer. We believe that the physiology of patients with cancer is not adequately represented in preclinical models, and the objective of this study was to determine how lung cancer will alter the nephrotoxicity of cisplatin. A genetically engineered mouse model and a syngeneic xenograft model of lung cancer were used. Mice were divided into the following four groups: 1) noncancer/vehicle, 2) noncancer/cisplatin, 3) cancer/vehicle, and 4) cancer/cisplatin. Mice were administered cisplatin via intraperitoneal injection once a week for 4 wk. Animals were euthanized 72 h following their final cisplatin injection. Mice with lung cancer had increased renal toxicity, injury, and fibrosis following repeated low doses of cisplatin. In addition, lung cancer alone induced kidney injury and fibrosis in the kidney before cisplatin treatment. In conclusion, this is the first study that we are aware of that assesses the impact of cancer on the kidney in conjunction with the nephrotoxicity of cisplatin. We believe that cancer is providing the first hit to the kidney and the subsequent damage from repeated doses of cisplatin becomes unsurmountable, leading to AKI and progression to chronic kidney disease.NEW & NOTEWORTHY Patients with cancer have impaired kidney function and increased susceptibility to nephrotoxic agents. Cisplatin is a commonly used chemotherapeutic with nephrotoxicity as the dose-limiting side effect. Cisplatin nephrotoxicity is almost exclusively studied in mice without cancer. Our current preclinical models do not adequately represent the complexity of patients with cancer. This study demonstrates increased renal toxicity, injury, and fibrosis in mice with lung cancer, which is exacerbated with cisplatin treatment. These results highlight the necessity of using preclinical models that more accurately capture the altered physiology of patients with cancer treated with cisplatin.

Case Report: Papilledema Secondary to Cerebral Venous Sinus Thrombosis after Severe COVID-19 Infection.

SIGNIFICANCE: This case highlights ocular adverse effects of a rare, potentially life-threatening complication from coronavirus disease 2019 (COVID-19). Papilledema can occur because of increased intracranial pressure caused by cerebral venous sinus thrombosis, the incidence of which may be more likely in patients with a history of COVID-19 because of an induced hypercoagulable state. PURPOSE: This case report presents a case of papilledema secondary to cerebral venous sinus thrombosis in a patient with a recent history of severe coronavirus disease (COVID-19). CASE REPORT: A 29-year-old man hospitalized with a complicated course of coronavirus disease (COVID-19) was referred to the ophthalmology department for episodic blurry vision of both eyes and intermittent binocular diplopia. Clinical examination revealed diffuse bilateral optic disc edema. Magnetic resonance venography of the brain during his admission revealed subtotal occlusion of the right transverse sinus by thrombosis. At the time of diagnosis, the patient was already taking systemic anticoagulation therapy for treatment of a recent pulmonary embolism also thought to be induced by COVID-19. After additional treatment with acetazolamide, there was improvement in his optic nerve edema. CONCLUSIONS: Cerebral venous sinus thrombosis, a serious and potentially life-threatening condition, can occur as a rare complication of COVID-19. In such cases, patients may develop increased intracranial pressure, papilledema, and subsequent vision loss. Magnetic resonance venography should be ordered in patients with suspected papilledema to help rule out the presence of cerebral venous sinus thrombosis.

Generating synthetic gait patterns based on benchmark datasets for controlling prosthetic legs.

BACKGROUND: Prosthetic legs help individuals with an amputation regain locomotion. Recently, deep neural network (DNN)-based control methods, which take advantage of the end-to-end learning capability of the network, have been proposed. One prominent challenge for these learning-based approaches is obtaining data for the training, particularly for the training of a mid-level controller. In this study, we propose a method for generating synthetic gait patterns (vertical load and lower limb joint angles) using a generative adversarial network (GAN). This approach enables a mid-level controller to execute ambulation modes that are not included in the training datasets. METHODS: The conditional GAN is trained on benchmark datasets that contain the gait data of individuals without amputation; synthetic gait patterns are generated from the user input. Further, a DNN-based controller for the generation of impedance parameters is trained using the synthetic gait pattern and the corresponding synthetic stiffness and damping coefficients. RESULTS: The trained GAN generated synthetic gait patterns with a coefficient of determination of 0.97 and a structural similarity index of 0.94 relative to benchmark data that were not included in the training datasets. We trained a DNN-based controller using the GAN-generated synthetic gait patterns for level-ground walking, standing-to-sitting motion, and sitting-to-standing motion. Four individuals without amputation participated in bypass testing and demonstrated the ambulation modes. The model successfully generated control parameters for the knee and ankle based on thigh angle and vertical load. CONCLUSIONS: This study demonstrates that synthetic gait patterns can be used to train DNN models for impedance control. We believe a conditional GAN trained on benchmark datasets can provide reliable gait data for ambulation modes that are not included in its training datasets. Thus, designing gait data using a conditional GAN could facilitate the efficient and effective training of controllers for prosthetic legs.

Wrist speed feedback improves elbow compensation and reaching accuracy for myoelectric transradial prosthesis users in hybrid virtual reaching task.

BACKGROUND: Myoelectric prostheses are a popular choice for restoring motor capability following the loss of a limb, but they do not provide direct feedback to the user about the movements of the device-in other words, kinesthesia. The outcomes of studies providing artificial sensory feedback are often influenced by the availability of incidental feedback. When subjects are blindfolded and disconnected from the prosthesis, artificial sensory feedback consistently improves control; however, when subjects wear a prosthesis and can see the task, benefits often deteriorate or become inconsistent. We theorize that providing artificial sensory feedback about prosthesis speed, which cannot be precisely estimated via vision, will improve the learning and control of a myoelectric prosthesis. METHODS: In this study, we test a joint-speed feedback system with six transradial amputee subjects to evaluate how it affects myoelectric control and adaptation behavior during a virtual reaching task. RESULTS: Our results showed that joint-speed feedback lowered reaching errors and compensatory movements during steady-state reaches. However, the same feedback provided no improvement when control was perturbed. CONCLUSIONS: These outcomes suggest that the benefit of joint speed feedback may be dependent on the complexity of the myoelectric control and the context of the task.

Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury.

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase-/-) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation-induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase-/- mice are resistant to cisplatin-induced AKI. nCDase-/- mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase-/- mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.

NFkB-signaling promotes glial reactivity and suppresses Müller glia-mediated neuron regeneration in the mammalian retina.

Müller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification of cell signaling pathways and gene regulatory networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here, we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however damage-induced NFkB activation is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGFß2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 signal transducer or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.

Microglial depletion abolishes ischemic preconditioning in white matter.

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.

Pharmacological inhibitors of autophagy have opposite effects in acute and chronic cisplatin-induced kidney injury.

The nephrotoxicity of cisplatin remains a major hurdle in the field of oncology. Thirty percent of patients treated with cisplatin develop acute kidney injury, and all patients are at risk for long-term impacts on kidney function. There are currently no Federal Drug Administration-approved agents to prevent or treat cisplatin-induced kidney injury. The dosing regimen used in preclinical models of nephrotoxicity may impact the success of therapeutic candidates in clinical trials. Here, we demonstrated that pharmacological inhibitors of autophagy have opposite effects when used as interventions in two different models of cisplatin-induced kidney injury. Eight-week-old male C57BL/6 mice were treated with either one dose of 20 mg/kg cisplatin or weekly doses of 9 mg/kg cisplatin for 4 wk or until body weight loss exceeded 30%. Concurrently, mice were administered multiple doses of 60 mg/kg chloroquine or 15 mg/kg 3-methyladenine attempting to globally inhibit autophagy. Mice that received a single high dose of cisplatin had worsened kidney function, inflammation, and cell death with the addition of chloroquine. 3-Methlyadenine did not impact the development of acute kidney injury in this model. In contrast, mice that received repeated low doses of cisplatin showed improved kidney function, reduced inflammation, and reduced fibrosis when treated with either chloroquine or 3-methyladenine. This study highlights how therapeutic candidates can have drastically different effects on the development of cisplatin-induced kidney injury depending on the dosing model used. This emphasizes the importance of choosing the appropriate model of injury for preclinical studies.NEW & NOTEWORTHY This study examined how inhibition of autophagy has opposite effects on the development of acute and chronic kidney injury. Autophagy inhibition exacerbated the development of acute kidney injury following a single high dose of cisplatin but prevented the development of injury and fibrosis following repeated low doses of cisplatin.

Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

A biological and chemical approach to restoring water quality: A case study in an urban eutrophic pond.

Efforts to improve water quality of eutrophic ponds often involve implementing changes to watershed management practices to reduce external nutrient loads. While this is required for long-term recovery and prevention, eutrophic conditions are often sustained through the recycling of internal nutrients already present within the waterbody. In particular, internal phosphorus bound to organic material and adsorbed to sediment has the potential to delay lake recovery for decades. Thus, pond and watershed management techniques are needed that not only reduce external nutrient loading but also mitigate the effects of internal nutrients already present. Therefore, our objective was to demonstrate a biological and chemical approach to remove and sequester nutrients present and entering an urban retention pond. A novel biological and chemical management technique was designed by constructing a 37 m2 (6.1 m × 6.1 m) floating treatment wetland coupled with a slow-release lanthanum composite inserted inside an airlift pump. The floating treatment wetland promoted microbial denitrification and plant uptake of nitrogen and phosphorus, while the airlift pump slowly released lanthanum to the water column over the growing season to reduce soluble reactive phosphorus. The design was tested at the microcosm and field scales, where nitrate-N and phosphate-P removal from the water column was significant (α = 0.05) at the microcosm scale and observed at the field scale. Two seasons of field sampling showed both nitrate-N and phosphate-P concentrations were reduced from 50 µg L-1 in 2020 to <10 µg L-1 in 2021. Load calculations of incoming nitrate-N and phosphate-P entering the retention pond from the surrounding watershed indicate the presented biological-chemical treatment is sustainable and will minimize the effects of nutrient loading from nonpoint source pollution.

Ubiquilin proteins regulate EGFR levels and activity in lung adenocarcinoma cells.

Ubiquilin (UBQLN) proteins are involved in diverse cellular processes like endoplasmic reticulum-associated degradation, autophagy, apoptosis, and epithelial-to-mesenchymal transition. UBQLNs interact with a variety of substrates, including cell surface receptors, transcription factor regulators, proteasomal machinery proteins, and transmembrane proteins. In addition, previous work from our lab shows that UBQLN1 interacts with insulin-like growth factor receptor family members (IGF1R, IGF2R, and INSR) and this interaction regulates the activity and proteostasis of IGFR family members. We wondered whether UBQLN proteins could also bind and regulate additional receptor tyrosine kinases. Thus, we investigated a link between UBQLN and the oncogene epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells. Loss of UBQLN1 occurs at high frequency in human lung cancer patient samples and we have shown that the loss of UBQLN1 is capable of altering processes involved in cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition in lung adenocarcinoma cell lines. Here, we present data that loss of UBQLN1 resulted in increased turnover of total EGFR while increasing the relative amount of phosphorylated EGFR in lung adenocarcinoma cells, especially in the presence of its ligand EGF. Furthermore, the loss of UBQLN1 led to a more invasive cell phenotype as manifested by increased proliferation, migration, and speed of movement of these lung adenocarcinoma cells. Taken together, UBQLN1 regulates the expression and stability of EGFR in lung cancer cells.

The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation.

Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis.

Coupling of shoulder joint torques in individuals with chronic stroke mirrors controls, with additional non-load-dependent negative effects in a combined-torque task.

BACKGROUND: After stroke, motor control is often negatively affected, leaving survivors with less muscle strength and coordination, increased tone, and abnormal synergies (coupled joint movements) in their affected upper extremity. Humeral internal and external rotation have been included in definitions of abnormal synergy but have yet to be studied in-depth. OBJECTIVE: Determine the ability to generate internal and external rotation torque under different shoulder abduction and adduction loads in persons with chronic stroke (paretic and non-paretic arm) and uninjured controls. METHODS: 24 participants, 12 with impairments after stroke and 12 controls, completed this study. A robotic device controlled abduction and adduction loading to 0, 25, and 50% of maximum strength in each direction. Once established against the vertical load, each participant generated maximum internal and external rotation torque in a dual-task paradigm. Four linear mixed-effects models tested the effect of group (control, non-paretic, and paretic), load (0, 25, 50% adduction or abduction), and their interaction on task performance; one model was created for each combination of dual-task directions (external or internal rotation during abduction or adduction). The protocol was then modeled using OpenSim to understand and explain the role of biomechanical (muscle action) constraints on task performance. RESULTS: Group was significant in all task combinations. Paretic arms were less able to generate internal and external rotation during abduction and adduction, respectively. There was a significant effect of load in three of four load/task combinations for all groups. Load-level and group interactions were not significant, indicating that abduction and adduction loading affected each group in a similar manner. OpenSim musculoskeletal modeling mirrored the experimental results of control and non-paretic arms and also, when adjusted for weakness, paretic arm performance. Simulations incorporating increased co-activation mirrored the drop in performance observed across all dual-tasks in paretic arms. CONCLUSION: Common biomechanical constraints (muscle actions) explain limitations in external and internal rotation strength during adduction and abduction dual-tasks, respectively. Additional non-load-dependent effects such as increased antagonist co-activation (hypertonia) may cause the observed decreased performance in individuals with stroke. The inclusion of external rotation in flexion synergy and of internal rotation in extension synergy may be over-simplifications.

Solution structure and functional investigation of human guanylate kinase reveals allosteric networking and a crucial role for the enzyme in cancer.

Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.

De novo synthesis of serine and glycine fuels purine nucleotide biosynthesis in human lung cancer tissues.

Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [13C6]glucose, D2-glycine, [13C2]glycine, and D3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro Surprisingly, [13C6]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D3-Ser was preferentially incorporated into purine rings over D2-glycine in both tissues and cell lines. MYC suppression attenuated [13C6]glucose, D3-serine, and [13C2]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy.

C57BL/6 mice require a higher dose of cisplatin to induce renal fibrosis and CCL2 correlates with cisplatin-induced kidney injury.

C57BL/6 mice are one of the most commonly used mouse strains in research, especially in kidney injury studies. However, C57BL/6 mice are resistant to chronic kidney disease-associated pathologies, particularly the development of glomerulosclerosis and interstitial fibrosis. Our laboratory and others developed a more clinically relevant dosing regimen of cisplatin (7 mg/kg cisplatin once a week for 4 wk and mice euthanized at day 24) that leads to the development of progressive kidney fibrosis in FVB/n mice. However, we found that treating C57BL/6 mice with this same dosing regimen does not result in kidney fibrosis. In this study, we demonstrated that increasing the dose of cisplatin to 9 mg/kg once a week for 4 wk is sufficient to consistently induce fibrosis in C57BL/6 mice while maintaining animal survival. In addition, we present that cohorts of C57BL/6 mice purchased from Jackson 1 yr apart and mice bred in-house display variability in renal outcomes following repeated low-dose cisplatin treatment. Indepth analyses of this intra-animal variability revealed C-C motif chemokine ligand 2 as a marker of cisplatin-induced kidney injury through correlation studies. In addition, significant immune cell infiltration was observed in the kidney after four doses of 9 mg/kg cisplatin, contrary to what has been previously reported. These results indicate that multiple strains of mice can be used with our repeated low-dose cisplatin model with dose optimization. Results also indicate that littermate control mice should be used with this model to account for population variability.