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Abstract Background Mesenchymal stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have demonstrated to elicit immunomodulatory and pro-regenerative properties that are beneficial for the treatment of chronic wounds. Thanks to different mediators, MSC-EVs have shown to play an important role in the proliferation, migration and cell survival of different skin cell populations. However, there is still a big bid to achieve the most effective, suitable and available source of MSC-EVs. Methods We isolated, characterized and compared medium-large EVs (m-lEVs) and small EVs (sEVs) obtained from hair follicle-derived MSCs (HF-MSCs) against the gold standard in regenerative medicine, EVs isolated from adipose tissue-derived MSCs (AT-MSCs). Results We demonstrated that HF-EVs, as well as AT-EVs, expressed typical MSC-EVs markers (CD9, CD44, CD63, CD81 and CD105) among other different functional markers. We showed that both cell types were able to increase human dermal fibroblasts (HDFs) proliferation and migration. Moreover, both MSC-EVs were able to increase angiogenesis in human umbilical vein endothelial cells (HUVECs) and protect HDFs exposed to a hyperglycemic environment from oxidative stress and cytotoxicity. Conclusions Taken together, HF-EVs demonstrated to exhibit comparable potential to that of AT-EVs as promising candidates in the treatment of chronic wounds.

Abstract Background Neurodegenerative diseases (NDs) are an accelerating global health problem. Nevertheless, the stronghold of the brain- the blood–brain barrier (BBB) prevents drug penetrance and dwindles effective treatments. Therefore, it is crucial to identify Trojan horse-like drug carriers that can effectively cross the blood–brain barrier and reach the brain tissue. We have previously developed polyunsaturated fatty acids (PUFA)-based nanostructured lipid carriers (NLC), namely DHAH-NLC. These carriers are modulated with BBB-permeating compounds such as chitosan (CS) and trans-activating transcriptional activator (TAT) from HIV-1 that can entrap neurotrophic factors (NTF) serving as nanocarriers for NDs treatment. Moreover, microglia are suggested as a key causative factor of the undergoing neuroinflammation of NDs. In this work, we used in vitro models to investigate whether DHAH-NLCs can enter the brain via the BBB and investigate the therapeutic effect of NTF-containing DHAH-NLC and DHAH-NLC itself on lipopolysaccharide-challenged microglia. Methods We employed human induced pluripotent stem cell-derived brain microvascular endothelial cells (BMECs) to capitalize on the in vivo-like TEER of this BBB model and quantitatively assessed the permeability of DHAH-NLCs. We also used the HMC3 microglia cell line to assess the therapeutic effect of NTF-containing DHAH-NLC upon LPS challenge. Results TAT-functionalized DHAH-NLCs successfully crossed the in vitro BBB model, which exhibited high transendothelial electrical resistance (TEER) values (≈3000 Ω*cm2). Specifically, the TAT-functionalized DHAH-NLCs showed a permeability of up to 0.4% of the dose. Furthermore, using human microglia (HMC3), we demonstrate that DHAH-NLCs successfully counteracted the inflammatory response in our cultures after LPS challenge. Moreover, the encapsulation of glial cell-derived neurotrophic factor (GNDF)-containing DHAH-NLCs (DHAH-NLC-GNDF) activated the Nrf2/HO-1 pathway, suggesting the triggering of the endogenous anti-oxidative system present in microglia. Conclusions Overall, this work shows that the TAT-functionalized DHAH-NLCs can cross the BBB, modulate immune responses, and serve as cargo carriers for growth factors; thus, constituting an attractive and promising novel drug delivery approach for the transport of therapeutics through the BBB into the brain.

Epistaxis is one of the most common otorhinolaryngology emergencies worldwide. Although there are currently several treatments available, they present several disadvantages. This, in addition to the increasing social need of being environmentally respectful, led us to investigate whether a sponge-like scaffold (SP–CH) produced from natural by-products of the food industry — soy protein and β-chitin — can be employed as a nasal pack for the treatment of epistaxis. To evaluate the potential of our material as a nasal pack, it was compared with two of the most commonly used nasal packs in the clinic: a basic gauze and the gold standard Merocel®. Our SP-CH presented great physicochemical and mechanical properties, lost weight in aqueous medium, and could even partially degrade when incubated in blood. It was shown to be both biocompatible and hemocompatible in vitro, clearing up any doubt about its safety. It showed increased blood clotting capacity in vitro, as well as increased capacity to bind both red blood cells and platelets, compared to the standard gauze and Merocel®. Finally, a rat-tail amputation model revealed that our SP-CH could even reduce bleeding time in vivo. This work, carried out from a circular economy approach, demonstrates that a green strategy can be followed to manufacture nasal packs using valorized by-products of the food industry, with equal or even better hemostatic properties than the gold standard in the clinic.

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.

Complex scaffolds composed of micro- and nano-structures are a key target in tissue engineering and the combination of sequential 3D printing and electrospinning enables the fabrication of these multi-scale structures. In this work, dual 3D printed and electrospun polycaprolactone (PCL) scaffolds with multiple mesh layers were successfully prepared. The scaffold macro- and micro-porosity were assessed by optical and scanning electron microscopy, showing that electrospun fibers formed aligned meshes within the pores of the scaffold. Consequently, the hydrophilicity of the scaffold increased with time, enhancing cell adhesion and growth. Additionally, compression tests in back and forth cycles demonstrated a good shape recovery behavior of the scaffolds. Biological results indicated that hybrid PCL scaffolds are biocompatible and enable a correct cell culture over time. Moreover, MC3T3-E1 preosteoblast culture on the scaffolds promoted the mineralization, increased the alkaline phosphatase (ALP) activity and upregulated the expression of early and late osteogenic markers, namely ALP and osteopontin (OPN), respectively. These results demonstrate that the sequential combination of 3D printing and electrospinning provides a facile method of incorporating fibers within a 3D printed scaffold, becoming a promising approach towards multi-scale hierarchical scaffolds capable of guiding the osteogenic differentiation.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been widely associated to beneficial effect over different neurodegenerative diseases. In the present study, we tested the potential therapeutic effect of docohexanoic acid (DHA) and its hydroxylated derivate, DHAH, in a partial lesion model of Parkinson's disease (PD). One month before and four months after the striatal lesion with 6-OHDA was made, the animals were daily treated with DHA (50 mg/kg), DHAH (50 mg/kg), vehicle or saline, by intragastric administration. Animal groups under n-3 PUFA treatments exhibited a trend to improve in amphetamine-induced rotations and cylinder test. The beneficial effect seen in behavioral studies were confirmed with TH immunostaining. TH+ fibers and TH+ neurons increased in the experimental groups treated with both n-3 PUFAs, DHA and DHAH. Moreover, the n-3 PUFAs administration decreased the astrogliosis and microgliosis, in both the striatum and substantia nigra (SN), with a higher decrease of GFAP+ and Iba-1+ cells for the DHAH treated group. This experimental group also revealed a positive effect on Nrf2 pathway regulation, decreasing the positive Nrf2 immmunostaining in the striatum and SN, which revealed a potential antioxidant effect of this compound. Taking together, these data suggest a positive effect of n-3 PUFAs administration, and more concretely of DHAH, for PD treatment as it exhibited positive results on dopaminergic system, neuroinflammation and oxidative stress.

Mesenchymal stromal cells (MSCs) have unique immunomodulatory capacities. We investigated hair follicle-derived MSCs (HF-MSCs) from the dermal sheath, which are advantageous as an alternative source because of their relatively painless and minimally risky extraction procedure. These cells expressed neural markers upon isolation and maintained stemness for a minimum of 10 passages. Furthermore, HF-MSCs showed responsiveness to pro-inflammatory environments by expressing type-II major histocompatibility complex antigens (MHC)-II to a lesser extent than adipose tissue-derived MSCs (AT-MSCs). HF-MSCs effectively inhibited the proliferation of peripheral blood mononuclear cells equivalently to AT-MSCs. Additionally, HF-MSCs promoted the induction of CD4+CD25+FOXP3+ regulatory T cells to the same extent as AT-MSCs. Finally, HF-MSCs, more so than AT-MSCs, skewed M0 and M1 macrophages towards M2 phenotypes, with upregulation of typical M2 markers CD163 and CD206 and downregulation of M1 markers such as CD64, CD86, and MHC-II. Thus, we conclude that HF-MSCs are a promising source for immunomodulation.

Considering the high prevalence and the complex pharmacological management of immune-mediated inflammatory diseases (IMIDs), the search for new therapeutic approaches for their treatment is vital. Although the immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSCs) have been extensively studied as a potential therapy in this field, direct MSC implantation presents some limitations that could slow down the clinical translation. Since the beneficial effects of MSCs have been mainly attributed to their ability to secrete a plethora of bioactive factors, their secretome has been proposed as a new and promising pathway for the treatment of IMIDs. Formed from soluble factors and extracellular vesicles (EVs), the MSC-derived secretome has been proven to elicit immunomodulatory effects that control the inflammatory processes that occur in IMIDs. This article aims to review the available knowledge on the MSC secretome, evaluating the advances in this field in terms of its composition, production and application, as well as analyzing the pending challenges in the field. Moreover, the latest research involving secretome administration in IMIDs is discussed to provide an updated state-of-the-art for this field. Finally, novel secretome delivery alternatives are reviewed, paying special attention to hydrogel encapsulation as one of the most convenient and promising strategies.

Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.

The aim of this review is to present 3D bioprinting of skin substitutes as an efficient approach of managing skin injuries. From a clinical point of view, classic treatments only provide physical protection from the environment, and existing engineered scaffolds, albeit acting as a physical support for cells, fail to overcome needs, such as neovascularisation. In the present work, the basic principles of bioprinting, together with the most popular approaches and choices of biomaterials for 3D-printed skin construct production, are explained, as well as the main advantages over other production methods. Moreover, the development of this technology is described in a chronological manner through examples of relevant experimental work in the last two decades: from the pioneers Lee et al. to the latest advances and different innovative strategies carried out lately to overcome the well-known challenges in tissue engineering of skin. In general, this technology has a huge potential to offer, although a multidisciplinary effort is required to optimise designs, biomaterials and production processes.

Protein-based conductive materials are gaining attention as alternative components of electronic devices for value-added applications. In this regard, soy protein isolate (SPI) was processed by extrusion in order to obtain SPI pellets, subsequently molded into SPI films by hot pressing, resulting in homogeneous and transparent films, as shown by scanning electron microscopy and UV-vis spectroscopy analyses, respectively. During processing, SPI denatured and refolded through intermolecular interactions with glycerol, causing a major exposition of tryptophan residues and fluorescence emission, affecting charge distribution and electron transport properties. Regarding electrical conductivity, the value found (9.889 × 10−4 S/m) is characteristic of electrical semiconductors, such as silicon, and higher than that found for other natural polymers. Additionally, the behavior of the films in contact with water was analyzed, indicating a controlled swelling and a hydrolytic surface, which is of great relevance for cell adhesion and spreading. In fact, cytotoxicity studies showed that the developed SPI films were biocompatible, according to the guidelines for the biological evaluation of medical devices. Therefore, these SPI films are uniquely suited as bioelectronics because they conduct both ionic and electronic currents, which is not accessible for the traditional metallic conductors.

Neurodegenerative diseases (ND) are one of the main problems of public health systems in the 21st century. The rise of nanotechnology-based drug delivery systems (DDS) has become in an emerging approach to target and treat these disorders related to the central nervous system (CNS). Among others, the use of nanostructured lipid carriers (NLCs) has increased in the last few years. Up to today, most of the developed NLCs have been made of a mixture of solid and liquid lipids without any active role in preventing or treating diseases. In this study, we successfully developed NLCs made of a functional lipid, such as the hydroxylated derivate of docohexaenoic acid (DHAH), named DHAH-NLCs. The newly developed nanocarriers were around 100 nm in size, with a polydispersity index (PDI) value of

Negatively charged microspheres (NCMs) represent a new therapeutic approach for wound healing since recent clinical trials have shown NCM efficacy in the recovery of hard-to-heal wounds that tend to stay in the inflammatory phase, unlocking the healing process. The aim of this study was to elucidate the NCM mechanism of action. NCMs were extracted from a commercial microsphere formulation (PolyHeal® Micro) and cytotoxicity, attachment, proliferation and viability assays were performed in keratinocytes and dermal fibroblasts, while macrophages were used for the phagocytosis and polarization assays. We demonstrated that cells tend to attach to the microsphere surface, and that NCMs are biocompatible and promote cell proliferation at specific concentrations (50 and 10 NCM/cell) by a minimum of 3 fold compared to the control group. Furthermore, NCM internalization by macrophages seemed to drive these cells to a noninflammatory condition, as demonstrated by the over-expression of CD206 and the under-expression of CD64, M2 and M1 markers, respectively. NCMs are an effective approach for reverting the chronic inflammatory state of stagnant wounds (such as diabetic wounds) and thus for improving wound healing.

In the current study, we developed a novel gelatin-based bilayer wound dressing. We used different crosslinking agents to confer unique properties to each layer, obtaining a bioinspired multifunctional hydrofilm suitable for wound healing. First, we produced a resistant and non-degradable upper layer by lactose-mediated crosslinking of gelatin, which provided mechanical support and protection to overall design. For the lower layer, we crosslinked gelatin with citric acid, resulting in a porous matrix with a great swelling ability. In addition, we incorporated chitosan into the lower layer to harness its wound healing ability. FTIR and SEM analyses showed that lactose addition changed the secondary structure of gelatin, leading to a more compact and smoother structure than that obtained with citric acid. The hydrofilm was able to swell 384.2 ± 57.2% of its dry weight while maintaining mechanical integrity. Besides, its water vapour transmission rate was in the range of commercial dressings (1381.5 ± 108.6 g/m2·day). In vitro, cytotoxicity assays revealed excellent biocompatibility. Finally, the hydrofilm was analysed through an ex vivo wound healing assay in human skin. It achieved similar results to the control in terms of biocompatibility and wound healing, showing suitable characteristics to be used as a wound dressing.

Background: The use of resveratrol as a dietary supplement is limited because it is easily oxidized and, after oral ingestion, it is metabolized into enterocytes and hepatocytes. Thus, new formulations are needed in order to improve its oral bioavailability. Objective: The objective of this study was to develop and characterize a gastro-resistant formulation of resveratrol for oral administration as a dietary supplement. Method: Resveratrol was encapsulated in Eudragit-coated pectin-alginate microparticles. Results: The microparticle size was about 1450 µm, with an encapsulation efficiency of 41.72% ± 1.92%. The dissolution assay conducted, as specified in the European Pharmacopoeia for delayed-release dosage forms, revealed that our microparticles were gastro-resistant, because the resveratrol percentage released from microparticles in acid medium was less than 10%. In addition, the high-performance liquid chromatographic (HPLC) method developed for resveratrol content quantification in the microparticles was validated according to International Council for Harmonisation (ICH) Q2 (R1) guidelines. Finally, the biological activity of resveratrol was investigated in 3T3-L1 mature adipocytes, concluding that the encapsulation process does not affect the activity of resveratrol. Conclusion: In summary, the gastro-resistant microparticles developed could represent a suitable method of including resveratrol in dietary supplements and in functional foods used in obesity therapy.

Abstract Background Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive, adult onset, non-inflammatory neuromuscular disorder with no effective treatment. The causative gene, GNE, codes for UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, which catalyzes the first two reactions in the synthesis of sialic acid. Reduced sialylation of muscle glycoproteins, such as α-dystroglycan and neural cell adhesion molecule (NCAM), has been reported in HIBM. Methods We treated 4 HIBM patients with intravenous immune globulin (IVIG), in order to provide sialic acid, because IgG contains 8 μmol of sialic acid/g. IVIG was infused as a loading dose of 1 g/kg on two consecutive days followed by 3 doses of 400 mg/kg at weekly intervals. Results For all four patients, mean quadriceps strength improved from 19.0 kg at baseline to 23.2 kg (+22%) directly after IVIG loading to 25.6 kg (+35%) at the end of the study. Mean shoulder strength improved from 4.1 kg at baseline to 5.9 kg (+44%) directly after IVIG loading to 6.0 kg (+46%) at the end of the study. The composite improvement for 8 other muscle groups was 5% after the initial loading and 19% by the end of the study. Esophageal motility and lingual strength improved in the patients with abnormal barium swallows. Objective measures of functional improvement gave variable results, but the patients experienced improvements in daily activities that they considered clinically significant. Immunohistochemical staining and immunoblotting of muscle biopsies for α-dystroglycan and NCAM did not provide consistent evidence for increased sialylation after IVIG treatment. Side effects were limited to transient headaches and vomiting. Conclusion The mild benefits in muscle strength experienced by HIBM patients after IVIG treatment may be related to the provision of sialic acid supplied by IVIG. Other sources of sialic acid are being explored as treatment options for HIBM.

Congenital facial weakness (CFW) encompasses a heterogenous set of rare disorders presenting with decreased facial movement from birth, secondary to impaired function of the facial musculature. The aim of the present study is to provide an analysis of subject-reported oral health-related quality of life (OHRQoL) in congenital facial weakness (CFW) disorders. Forty-four subjects with CFW and age- and sex- matched controls were enrolled in an Institutional Review Board (IRB)-approved study. Demographic data, medical and surgical history, comprehensive oral examination, and the Oral Health Impact Profile (OHIP-14) were obtained. Compared to unaffected controls, subjects with CFW had higher OHIP-14 scores overall (mean ± SD: 13.11 ± 8.11 vs. 4.46 ± 4.98, p < 0.0001) and within five of seven oral health domains, indicating decreased OHRQoL. Although subjects with Moebius syndrome (MBS) were noted to have higher OHIP-14 scores than those with Hereditary Congenital Facial Paresis (HCFP), there was no significant correlation in OHIP-14 score to age, sex, or specific diagnosis. An increase in OHIP-14 scores in subjects was detected in those who had undergone reanimation surgery. In conclusion, subjects with CFW had poorer OHRQoL compared to controls, and subjects with MBS had poorer OHRQoL than subjects with HCFP. This study provides better understanding of oral health care needs and quality of life in a CFW cohort and suggests that guidelines for dental treatment are required.

Propionic acidemia (PA) is an autosomal recessive condition (OMIM #606054), wherein pathogenic variants in PCCA and PCCB impair the activity of propionyl-CoA carboxylase. PA is associated with neurodevelopmental disorders, including intellectual disability (ID) and autism spectrum disorder (ASD); however, the correlates and mechanisms of these outcomes remain unknown. Using data from a subset of participants with PA enrolled in a dedicated natural history study (n = 33), we explored associations between neurodevelopmental phenotypes and laboratory parameters. Twenty (61%) participants received an ID diagnosis, and 12 of the 31 (39%) who were fully evaluated received the diagnosis of ASD. A diagnosis of ID, lower full-scale IQ (sample mean = 65 ± 26), and lower adaptive behavior composite scores (sample mean = 67 ± 23) were associated with several biomarkers. Higher concentrations of plasma propionylcarnitine, plasma total 2-methylcitrate, serum erythropoietin, and mitochondrial biomarkers plasma FGF21 and GDF15 were associated with a more severe ID profile. Reduced 1- 13 C-propionate oxidative capacity and decreased levels of plasma and urinary glutamine were also associated with a more severe ID profile. Only two parameters, increased serum erythropoietin and decreased plasma glutamine, were associated with ASD. Plasma glycine, one of the defining features of PA, was not meaningfully associated with either ID or ASD. Thus, while both ID and ASD were commonly observed in our PA cohort, only ID was robustly associated with metabolic parameters. Our results suggest that disease severity and associated mitochondrial dysfunction may play a role in CNS complications of PA and identify potential biomarkers and candidate surrogate endpoints., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

A distinct adipose tissue distribution pattern was observed in patients with methylmalonyl-CoA mutase deficiency, an inborn error of branched-chain amino acid (BCAA) metabolism, characterized by centripetal obesity with proximal upper and lower extremity fat deposition and paucity of visceral fat, that resembles familial multiple lipomatosis syndrome. To explore brown and white fat physiology in methylmalonic acidemia (MMA), body composition, adipokines, and inflammatory markers were assessed in 46 patients with MMA and 99 matched controls. Fibroblast growth factor 21 levels were associated with acyl-CoA accretion, aberrant methylmalonylation in adipose tissue, and an attenuated inflammatory cytokine profile. In parallel, brown and white fat were examined in a liver-specific transgenic MMA mouse model (Mmut-/- TgINS-Alb-Mmut). The MMA mice exhibited abnormal nonshivering thermogenesis with whitened brown fat and had an ineffective transcriptional response to cold stress. Treatment of the MMA mice with bezafibrates led to clinical improvement with beiging of subcutaneous fat depots, which resembled the distribution seen in the patients. These studies defined what we believe to be a novel lipodystrophy phenotype in patients with defects in the terminal steps of BCAA oxidation and demonstrated that beiging of subcutaneous adipose tissue in MMA could readily be induced with small molecules.

Humans rely heavily on facial expressions for social communication to convey their thoughts and emotions and to understand them in others. One prominent but controversial view is that humans learn to recognize the significance of facial expressions by mimicking the expressions of others. This view predicts that an inability to make facial expressions (e.g., facial paralysis) would result in reduced perceptual sensitivity to others' facial expressions. To test this hypothesis, we developed a diverse battery of sensitive emotion recognition tasks to characterize expression perception in individuals with Moebius Syndrome (MBS), a congenital neurological disorder that causes facial palsy. Using computer-based detection tasks we systematically assessed expression perception thresholds for static and dynamic face and body expressions. We found that while MBS individuals were able to perform challenging perceptual control tasks and body expression tasks, they were less efficient at extracting emotion from facial expressions, compared to matched controls. Exploratory analyses of fMRI data from a small group of MBS participants suggested potentially reduced engagement of the amygdala in MBS participants during expression processing relative to matched controls. Collectively, these results suggest a role for facial mimicry and consequent facial feedback and motor experience in the perception of others' facial expressions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Published by Elsevier Ltd.)

Hereditary congenital facial paresis type 1 (HCFP1) is an autosomal dominant disorder of absent or limited facial movement that maps to chromosome 3q21-q22 and is hypothesized to result from facial branchial motor neuron (FBMN) maldevelopment. In the present study, we report that HCFP1 results from heterozygous duplications within a neuron-specific GATA2 regulatory region that includes two enhancers and one silencer, and from noncoding single-nucleotide variants (SNVs) within the silencer. Some SNVs impair binding of NR2F1 to the silencer in vitro and in vivo and attenuate in vivo enhancer reporter expression in FBMNs. Gata2 and its effector Gata3 are essential for inner-ear efferent neuron (IEE) but not FBMN development. A humanized HCFP1 mouse model extends Gata2 expression, favors the formation of IEEs over FBMNs and is rescued by conditional loss of Gata3. These findings highlight the importance of temporal gene regulation in development and of noncoding variation in rare mendelian disease., (© 2023. The Author(s).)

Methylmalonic Acidemia (MMA) is a heterogenous group of inborn errors of metabolism caused by a defect in the methylmalonyl-CoA mutase (MMUT) enzyme or the synthesis and transport of its cofactor, 5'-deoxy-adenosylcobalamin. It is characterized by life-threatening episodes of ketoacidosis, chronic kidney disease, and other multiorgan complications. Liver transplantation can improve patient stability and survival and thus provides clinical and biochemical benchmarks for the development of hepatocyte-targeted genomic therapies. Data are presented from a US natural history protocol that evaluated subjects with different types of MMA including mut-type (N = 91), cblB-type (15), and cblA-type MMA (17), as well as from an Italian cohort of mut-type (N = 19) and cblB-type MMA (N = 2) subjects, including data before and after organ transplantation in both cohorts. Canonical metabolic markers, such as serum methylmalonic acid and propionylcarnitine, are variable and affected by dietary intake and renal function. We have therefore explored the use of the 1- 13 C-propionate oxidation breath test (POBT) to measure metabolic capacity and the changes in circulating proteins to assess mitochondrial dysfunction (fibroblast growth factor 21 [FGF21] and growth differentiation factor 15 [GDF15]) and kidney injury (lipocalin-2 [LCN2]). Biomarker concentrations are higher in patients with the severe mut 0 -type and cblB-type MMA, correlate with a decreased POBT, and show a significant response postliver transplant. Additional circulating and imaging markers to assess disease burden are necessary to monitor disease progression. A combination of biomarkers reflecting disease severity and multisystem involvement will be needed to help stratify patients for clinical trials and assess the efficacy of new therapies for MMA., (© 2023 SSIEM. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase ( MMUT ) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of ∼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9- Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation., Competing Interests: R.J.C., G.D., J.A.C., and C.P.V. are inventors on a patent application filed by NIH on their behalf on the use of AAV44.9 as a gene therapy vector to treat MMA., (© 2022.)

Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses, and improve patient care for patients with rare conditions., (© 2022 SSIEM. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)