Evaluation of the clinical, biochemical, and molecular spectrum of Cobalamin C (CblC) defect in 33 patients from Pakistan.

BACKGROUND: Cobalamin C is the most common inborn error of intracellular cobalamin metabolism caused by biallelic pathogenic variants in the MMACHC gene, leading to impaired conversion of dietary vitamin B12 into its two metabolically active forms, methylcobalamin and adenosylcobalamin. Biochemical hallmarks are elevated plasma total homocysteine (HCYs) and low methionine accompanied by methylmalonic aciduria. This study aimed to evaluate the clinical, biochemical, and molecular analysis of Pakistani patients with CblC defect. METHODS: Medical charts, urine organic acid (UOA) chromatograms, plasma amino acid levels, plasma tHcy and MMACHC gene results of patients presenting at the Biochemical Genetics Clinic, AKUH from 2013-2021 were reviewed. Details were collected on a pre-structured questionnaire. SPSS 22 was used for data analysis. RESULTS: CblC was found in 33 cases (Male:Female 19:14). The median age of symptoms onset and diagnosis were 300 (IQR:135-1800) and 1380 (IQR: 240-2730) days. The most common clinical features were cognitive impairment (n = 29), seizures (n = 23), motor developmental delay (n = 20), hypotonia (n = 17), and sparse/hypopigmented scalp hair (n = 16). The MMACHC gene sequencing revealed homozygous pathogenic variant c.394C > T, (p.Arg132*) in 32 patients, whereas c.609G > A, (p.TRP203*) in one patient whose ancestors had settled in Pakistan from China decades ago. The median age of treatment initiation was 1530 (IQR: 240-2790). The median pre-treatment HCYs levels were 134 (IQR:87.2-155.5) compared to post-treatment levels of 33.3 (IQR: 27.3-44.95) umol/L. CONCLUSIONS: Thirty-three cases of CblC defect from a single center underscores a significant number of the disorder within Pakistan. Late diagnosis emphasizes the need for increased clinical awareness and adequate diagnostic facilities.

Cortical bone plate properties assessment using inversion of axially transmitted low frequency ultrasonic guided waves.

Over the past few decades, early osteoporosis detection using ultrasonic bone quality evaluation has gained prominence. Specifically, various studies focused on axial transmission using ultrasonic guided waves and have highlighted this technique's sensitivity to intrinsic properties of long cortical bones. This work aims to demonstrate the potential of low-frequency ultrasonic guided waves to infer the properties of the bone inside which they are propagating. A proprietary ultrasonic transducer, tailored to transmit ultrasonic guided waves under 500 kHz, was used for the data collection. The gathered data underwent two-dimensional fast Fourier transform processing to extract experimental dispersion curves. The proposed inversion scheme compares experimental dispersion curves with simulated dispersion curves calculated through the semi-analytical iso-geometric analysis (SAIGA) method. The numerical model integrates a bone phantom plate coupled with a soft tissue layer on its top surface, mimicking the experimental bone phantom plates. Subsequently, the mechanical properties of the bone phantom plates were estimated by reducing the misfit between the experimental and simulated dispersion curves. This inversion leaned heavily on the dispersive trajectories and amplitudes of ultrasonic guided wave modes. Results indicate a marginal discrepancy under 5% between the mechanical properties ascertained using the SAIGA-based inversion and those measured using bulk wave pulse-echo measurements.

Mitochondrial complex I promotes kidney cancer metastasis.

Most kidney cancers are metabolically dysfunctional1-4, but how this dysfunction affects cancer progression in humans is unknown. We infused 13C-labelled nutrients in over 80 patients with kidney cancer during surgical tumour resection. Labelling from [U-13C]glucose varies across subtypes, indicating that the kidney environment alone cannot account for all tumour metabolic reprogramming. Compared with the adjacent kidney, clear cell renal cell carcinomas (ccRCCs) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in ex vivo organotypic cultures, indicating that suppressed labelling is tissue intrinsic. [1,2-13C]acetate and [U-13C]glutamine infusions in patients, coupled with measurements of respiration in isolated human kidney and tumour mitochondria, reveal lower electron transport chain activity in ccRCCs that contributes to decreased oxidative and enhanced reductive TCA cycle labelling. However, ccRCC metastases unexpectedly have enhanced TCA cycle labelling compared with that of primary ccRCCs, indicating a divergent metabolic program during metastasis in patients. In mice, stimulating respiration or NADH recycling in kidney cancer cells is sufficient to promote metastasis, whereas inhibiting electron transport chain complex I decreases metastasis. These findings in humans and mice indicate that metabolic properties and liabilities evolve during kidney cancer progression, and that mitochondrial function is limiting for metastasis but not growth at the original site.

Niacin supplementation in a child with novel MTTN variant m.5670A>G causing early onset mitochondrial myopathy and NAD+ deficiency.

Myopathy is a common manifestation in mitochondrial disorders, but the pathomechanisms are still insufficiently studied in children. Here, we report a severe, progressive mitochondrial myopathy in a four-year-old child, who died at eight years. He developed progressive loss of muscle strength with nocturnal hypoventilation and dilated cardiomyopathy. Skeletal muscle showed ragged red fibers and severe combined respiratory chain deficiency. Mitochondrial DNA sequencing revealed a novel m.5670A>G mutation in mitochondrial tRNAAsn (MTTN) with 88 % heteroplasmy in muscle. The proband also had systemic NAD+ deficiency but rescuing this with the NAD+ precursor niacin did not stop disease progression. Targeted metabolomics revealed an overall shift of metabolism towards controls after niacin supplementation, with normalized tryptophan metabolites and lipid-metabolic markers, but most amino acids did not respond to niacin therapy. To conclude, we report a new MTTN mutation, secondary NAD+ deficiency in childhood-onset mitochondrial myopathy with metabolic but meager clinical response to niacin supplementation.

Millimeter-scale soft capsules for sampling liquids in fluid-filled confined spaces.

Sampling liquids in small and confined spaces to retrieve chemicals and microbiomes could enable minimally invasive monitoring human physiological conditions for understanding disease development and allowing early screening. However, existing tools are either invasive or too large for sampling liquids in tortuous and narrow spaces. Here we report a fundamental liquid sampling mechanism that enables millimeter-scale soft capsules for sampling liquids in confined spaces. The miniature capsule is enabled by flexible magnetic valves and superabsorbent polymer, fully wirelessly controlled for on-demand fluid sampling. A group of miniature capsules could navigate in fluid-filled and confined spaces safely using a rolling locomotion. The integration of on-demand triggering, sampling, and sealing mechanism and the agile group locomotion allows us to demonstrate precise control of the soft capsules, navigating and sampling body fluids in a phantom and animal organ ex vivo, guided by ultrasound and x-ray medical imaging. The proposed mechanism and wirelessly controlled devices spur the next-generation technologies for minimally invasive disease diagnosis.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage.

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

3-D finite element model of the impaction of a press-fitted femoral stem under various biomechanical environments.

BACKGROUND: Uncemented femoral stem insertion into the bone is achieved by applying successive impacts on an inserter tool called "ancillary". Impact analysis has shown to be a promising technique to monitor the implant insertion and to improve its primary stability. METHOD: This study aims to provide a better understanding of the dynamic phenomena occurring between the hammer, the ancillary, the implant and the bone during femoral stem insertion, to validate the use of impact analyses for implant insertion monitoring. A dynamic 3-D finite element model of the femoral stem insertion via an impaction protocol is proposed. The influence of the trabecular bone Young's modulus (Et), the interference fit (IF), the friction coefficient at the bone-implant interface (µ) and the impact velocity (v0) on the implant insertion and on the impact force signal is evaluated. RESULTS: For all configurations, a decrease of the time difference between the two first peaks of the impact force signal is observed throughout the femoral stem insertion, up to a threshold value of 0.23 ms. The number of impacts required to reach this value depends on Et, v0 and IF and varies between 3 and 8 for the set of parameters considered herein. The bone-implant contact ratio reached after ten impacts varies between 60% and 98%, increases as a function of v0 and decreases as a function of IF, µ and Et. CONCLUSION: This study confirms the potential of an impact analyses-based method to monitor implant insertion and to retrieve bone-implant contact properties.

Characterization of the concentration of agar-based soft tissue mimicking phantoms by impact analysis.

In various medical fields, a change of soft tissue stiffness is associated with its physio-pathological evolution. While elastography is extensively employed to assess soft tissue stiffness in vivo, its application requires a complex and expensive technology. The aim of this study is to determine whether an easy-to-use method based on impact analysis can be employed to determine the concentration of agar-based soft tissue mimicking phantoms. Impact analysis was performed on soft tissue mimicking phantoms made of agar gel with a mass concentration ranging from 1% to 5%. An indicator Δt is derived from the temporal variation of the impact force signal between the hammer and a small beam in contact with the sample. The results show a non-linear decrease of Δt as a function of the agar concentration (and thus of the sample stiffness). The value of Δt provides an estimation of the agar concentration with an error of 0.11%. This sensitivity of the impact analysis based method to the agar concentration is of the same order of magnitude than results obtained with elastography techniques. This study opens new paths towards the development of impact analysis for a fast, easy and relatively inexpensive clinical evaluation of soft tissue elastic properties.

New insights in the targets of action of dimethyl fumarate in endothelial cells: effects on energetic metabolism and serine synthesis in vitro and in vivo.

Dimethyl fumarate is an ester from the Krebs cycle intermediate fumarate. This drug is approved and currently used for the treatment of psoriasis and multiple sclerosis, and its anti-angiogenic activity was reported some years ago. Due to the current clinical relevance of this compound and the recently manifested importance of endothelial cell metabolism on the angiogenic switch, we wanted to elucidate whether dimethyl fumarate has an effect on energetic metabolism of endothelial cells. Different experimental approximations were performed in endothelial cells, including proteomics, isotope tracing and metabolomics experimental approaches, in this work we studied the possible role of dimethyl fumarate in endothelial cell energetic metabolism. We demonstrate for the first time that dimethyl fumarate promotes glycolysis and diminishes cell respiration in endothelial cells, which could be a consequence of a down-regulation of serine and glycine synthesis through inhibition of PHGDH activity in these cells. Dimethyl fumarate alters the energetic metabolism of endothelial cells in vitro and in vivo through an unknown mechanism, which could be the cause or the consequence of its pharmacological activity. This new discovery on the targets of this compound could open a new field of study regarding the mechanism of action of dimethyl fumarate.

Adipocytes reprogram cancer cell metabolism by diverting glucose towards glycerol-3-phosphate thereby promoting metastasis.

In the tumor microenvironment, adipocytes function as an alternate fuel source for cancer cells. However, whether adipocytes influence macromolecular biosynthesis in cancer cells is unknown. Here we systematically characterized the bidirectional interaction between primary human adipocytes and ovarian cancer (OvCa) cells using multi-platform metabolomics, imaging mass spectrometry, isotope tracing and gene expression analysis. We report that, in OvCa cells co-cultured with adipocytes and in metastatic tumors, a part of the glucose from glycolysis is utilized for the biosynthesis of glycerol-3-phosphate (G3P). Normoxic HIF1α protein regulates the altered flow of glucose-derived carbons in cancer cells, resulting in increased glycerophospholipids and triacylglycerol synthesis. The knockdown of HIF1α or G3P acyltransferase 3 (a regulatory enzyme of glycerophospholipid synthesis) reduced metastasis in xenograft models of OvCa. In summary, we show that, in an adipose-rich tumor microenvironment, cancer cells generate G3P as a precursor for critical membrane and signaling components, thereby promoting metastasis. Targeting biosynthetic processes specific to adipose-rich tumor microenvironments might be an effective strategy against metastasis.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage.

Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. While many studies have documented the importance of mitochondrial oxidative phosphorylation (OXPHOS) in tumor growth, some cancer cells experience conditions of reduced OXPHOS in vivo and induce alternative metabolic pathways to compensate. To assess how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts and plasma from patients with inborn errors of mitochondrial metabolism, and in cancer cells subjected to inhibition of the electron transport chain (ETC). All these analyses revealed extensive perturbations in purine-related metabolites; in non-small cell lung cancer (NSCLC) cells, ETC blockade led to purine metabolite accumulation arising from a reduced cytosolic NAD + /NADH ratio (NADH reductive stress). Stable isotope tracing demonstrated that ETC deficiency suppressed de novo purine nucleotide synthesis while enhancing purine salvage. Analysis of NSCLC patients infused with [U- 13 C]glucose revealed that tumors with markers of low oxidative mitochondrial metabolism exhibited high expression of the purine salvage enzyme HPRT1 and abundant levels of the HPRT1 product inosine monophosphate (IMP). ETC blockade also induced production of ribose-5' phosphate (R5P) by the pentose phosphate pathway (PPP) and import of purine nucleobases. Blocking either HPRT1 or nucleoside transporters sensitized cancer cells to ETC inhibition, and overexpressing nucleoside transporters was sufficient to drive growth of NSCLC xenografts. Collectively, this study mechanistically delineates how cells compensate for suppressed purine metabolism in response to ETC blockade, and uncovers a new metabolic vulnerability in tumors experiencing NADH excess.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

Correction: Vu et al. Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance. Metabolites 2022, 12, 385.

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L-2-hydroxyglutaric aciduria - review of literature and case series.

L-2-hydroxyglutaric aciduria (L2HGA) is an autosomal recessive, slowly progressive neurodegenerative disease characterized by psychomotor delay and cerebellar dysfunction. The biochemical hallmark is increased concentrations of L2HG in body fluids. Brain MRI exhibits characteristic centripetal extension of the white matter involvement that differentiates it from other leukodystrophies. The authors report two sisters from Pakistan with L2HGA with 4 years of follow-up. The authors have also compared the clinical outcome of our patients with 45 previously reported patients with L2HGA for whom treatment and clinical outcome was reported. Case presentation: The authors report two sisters with L2HGA from Pakistan born to consanguineous parents. The 15- and 17-year-old girls presented with psychomotor delay, seizures, ataxia, intentional tremors, and dysarthria. Both had normal anthropometric measurements for age. Exaggerated tendon reflexes and bilateral sustained ankle clonus were observed in addition to cerebellar signs. Urine organic acids analysis showed marked excretion of 2-hydroxyglutaric acid, chiral differentiation of 2-hydroxyglutaric acid showed it to be L2HGA. Brain MRI of the 15-year-old showed diffuse subcortical white matter changes evident by T2/FLAIR hyperintense signals bilaterally, particularly in the frontal region in the centripetal distribution with some diffusion restriction along involvement of globus pallidus. The characteristic MRI pattern raised the suspicion of L2HGA. Targeted L2HGDH sequencing identified a homozygous pathogenic variant, c.829C>T (p.Arg227*) in L2HGDH gene in both girls. Both parents were heterozygous carriers of the familial variant. Conclusion: Neuroradiological features of centripetal subcortical leukoencephalopathy with basal ganglia and dentate nuclei involvement are rather specific to L2HGA and should lead to further biochemical investigations to look for L2HGA and L2HGDH gene sequencing.

Genomic characterization of endemic diarrheagenic Escherichia coli and Escherichia albertii from infants with diarrhea in Vietnam.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) is a group of bacterial pathogens that causes life-threatening diarrhea in children in developing countries. However, there is limited information on the characteristics of DEC isolated from patients in these countries. A detailed genomic analysis of 61 DEC-like isolates from infants with diarrhea was performed to clarify and share the characteristics of DEC prevalent in Vietnam. PRINCIPAL FINDINGS: DEC was classified into 57 strains, including 33 enteroaggregative E. coli (EAEC) (54.1%), 20 enteropathogenic E. coli (EPEC) (32.8%), two enteroinvasive E. coli (EIEC) (3.3%), one enterotoxigenic E. coli (ETEC), and one ETEC/EIEC hybrid (1.6% each), and surprisingly into four Escherichia albertii strains (6.6%). Furthermore, several epidemic DEC clones showed an uncommon combination of pathotypes and serotypes, such as EAEC Og130:Hg27, EAEC OgGp9:Hg18, EAEC OgX13:H27, EPEC OgGp7:Hg16, and E. albertii EAOg1:HgUT. Genomic analysis also revealed the presence of various genes and mutations associated with antibiotic resistance in many isolates. Strains that demonstrate potential resistance to ciprofloxacin and ceftriaxone, drugs recommended for treating childhood diarrhea, accounted for 65.6% and 41%, respectively. SIGNIFICANCE: Our finding indicate that the routine use of these antibiotics has selected resistant DECs, resulting in a situation where these drugs do not provide in therapeutic effects for some patients. Bridging this gap requires continuous investigations and information sharing regarding the type and distribution of endemic DEC and E. albertii and their antibiotic resistance in different countries.

Recessive pathogenic variants in MCAT cause combined oxidative phosphorylation deficiency.

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, including Mcat, is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants in MCAT. Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Re-expression of wild-type MCAT rescued the phenotype in patient fibroblasts. This is the first report of a patient with MCAT pathogenic variants and combined oxidative phosphorylation deficiency.

Detection of periprosthetic fractures around the femoral stem by resonance frequency analysis: An in vitro study.

Periprosthetic femoral bone fractures are frequent complications of Total Hip Arthroplasty (THA) and may occur during the insertion of uncemented Femoral Stems (FS), due to the nature of the press-fit fixation. Such fracture may lead to the surgical failure of the THA and require a revision surgery, which may have dramatic consequences. Therefore, an early detection of intra-operative fractures is important to avoid worsening the fracture and/or to enable a peroperative treatment. The aim of this in vitro study is to determine the sensitivity of a method based on resonance frequency analysis of the bone-stem-ancillary system for periprosthetic fractures detection. A periprosthetic fracture was artificially created close to the lesser-trochanter of 10 femoral bone mimicking phantoms. The bone-stem-ancillary resonance frequencies in the range (2-12) kHz were measured on an ancillary instrumented with piezoelectric sensors, which was fixed to the femoral stem. The measurements were repeated for different fracture lengths from 4 to 55 mm. The results show a decrease of the resonance frequencies due to the fracture occurrence and propagation. The frequency shift reached up to 170 Hz. The minimum fracture length that can be detected varies from 3.1±1.7 mm to 5.9±1.9 mm according to the mode and to the specimen. A significantly higher sensitivity (p = 0.011) was obtained for a resonance frequency around 10.6 kHz, corresponding to a mode vibrating in a plane perpendicular to the fracture. This study opens new paths toward the development of non-invasive vibration-based methods for intra-operative periprosthetic fractures detection.