In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds

In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.     

Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice

X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials.X-linked myotubular myopathy (XLMTM) is a severe form of congenital myopathy with an estimated incidence of 1:50,000 male births that most often presents with severe perinatal weakness and respiratory failure.^1,2 Many patients with XLMTM die within the first year of life despite the use of mechanical ventilation, and no treatments approved by the Food and Drug Administration are available. XLMTM is caused by mutations in the gene that encodes myotubularin (MTM1), which is a phosphoinositide phosphatase thought to be involved in endosomal trafficking, cytoskeletal organization, apoptosis, and/or maintenance of the sarcoplasmic reticulum/T-tubular system within myofibers.^3–8 Muscle biopsies from patients with XLMTM display excessively small fibers with increased numbers of fibers that contain central nuclei and central aggregation of organelles.⁹ Although the number of centrally nucleated fibers bears little relationship to a patient''s prognosis, there is a clear correlation between the degree of fiber smallness at birth and the severity of the patients'' disease.¹⁰ Two murine models of myotubularin deficiency are used, the severely symptomatic Mtm1δ4 (also referred to as Mtm1 knockout in prior studies^3,11,12) and the moderately symptomatic Mtm1 p.R69C mice,¹³ both of which display weakness and myofiber smallness and similar pathology to that seen in XLMTM.Because of the relationship between myofiber size and symptomatic severity in patients with XLMTM and in Mtm1δ4 mice, we had previously hypothesized that correction of myofiber smallness in myotubularin deficiency would greatly improve strength. Inhibitors of myostatin or nonfunctional decoys of its receptor, the activin type IIB receptor (ActRIIB), can be used to inhibit this negative regulator of myofiber size, leading to myofiber hypertrophy. Myostatin binds to (and signals through) the ActRIIB to activate the transforming growth factor-β pathway, which prevents progression through the cell cycle and down-regulates several key processes related to myofiber hypertrophy.^14,15 We recently reported a trial of ActRIIB-mFC in Mtm1δ4 mice, which produced 17% extension of life span, with transient increases in weight, forelimb grip strength, myofiber size, and myofiber hypertrophy restricted to type 2b myofibers in Mtm1δ4 animals.¹² Interestingly, ActRIIB-mFc produces hypertrophy in all muscle fiber types in wild-type (WT) mice,^12,16 which suggests that myotubularin deficiency interferes with the activation of hypertrophic pathways in oxidative fibers.We hypothesized that the transience of the therapeutic effects observed in treated Mtm1δ4 mice may have been related to the severity of the disease, so we have now repeated this study in the less severely affected Mtm1 p.R69C mouse.¹³ Surprisingly, treatment of Mtm1 p.R69C mice did not produce significant increases in animal weight or grip strength, and treatment-induced myofiber hypertrophy was only observed in the Mtm1 p.R69C gastrocnemius muscles. The ability of these muscles to respond to ActRIIB-mFC treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. The main difference between treatment-responsive (gastrocnemius) and treatment-resistant (quadriceps) muscles in Mtm1 p.R69C mice was related to low levels of phosphorylated ribosomal protein 6 (p-rpS6) and high levels of eukaryotic elongation factor 2 kinase (eEF2K) in the treatment-responsive gastrocnemius muscle that were not observed in other Mtm1 p.R69C muscles or in WT mice. rpS6 and eEF2K are terminal signaling molecules of the insulinlike growth factor-1/Akt and extracellular signal-related kinase (ERK) pathways that are involved in the fine-tuning of global protein synthesis, with a role in the determination of cell size that remains unclear (reviewed in Meyuhas¹⁷). Our findings indicate that the response to hypertrophic agents does not always correlate with activities of known hypertrophic pathways, such as the Akt pathway, but unexpectedly varies both by muscle type and fiber type and in XLMTM is affected by the nature of the Mtm1 mutation. These results highlight that there is much we still do not understand about the control of muscle size and emphasize the importance of evaluating multiple muscle and fiber types in future trials of hypertrophic therapies.     

Genetic and Epigenetic Determinants of Low Dysferlin Expression in Monocytes

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle, and peripheral blood monocytes. Expression in skeletal muscle and monocytes strongly correlates in healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While 18 of these individuals with protein levels in the range of 40%–64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of 18 detected missense variants in only four. Analysis of DNA methylation patterns at the DYSF locus showed no changes in methylation levels at CpG islands and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, the monocyte assay cannot be used to determine the carrier status; for this, a molecular analysis of DYSF must be performed.     

Thyrolipoma and thyrolipomatosis: 5 case reports and historical review of the literature

Because thyrolipoma (adenolipoma of thyroid) and thyrolipomatosis (diffuse lipomatosis of thyroid) are distinctively rare conditions with only few cases reported in the literature, we are reporting 5 additional cases. All the 5 patients were adult females, with ages from 38 to 79 years, who presented with thyroid masses. Four of the patients had normal thyroid function tests and one had mild hypothyroidism. All patients received partial or total thyroidectomy. The thyroid specimens showed either circumscribed yellow-tan masses (cases 1, 2, and 3) or diffuse yellow-brown discoloration (cases 4 and 5). Histologic examination revealed abundant mature fat infiltrating the affected thyroid tissue in 3 distinct patterns: (1) fat infiltration limited to follicular adenomas (thyrolipoma); (2) fat diffusely infiltrating throughout the thyroid gland (thyrolipomatosis); or (3) fat infiltration involving both follicular adenoma and their surrounding thyroid tissue. Because of the rarity of thyroid fat-containing lesions, confusion in differential diagnosis may occasionally occur. It is important to be aware during frozen section that these lesions may present as extrathyroidal nodules, which can be radioactive on intraoperative scan for parathyroid glands. In addition, a papillary thyroid carcinoma was also identified in one case of thyrolipomatosis. All patients recovered well after surgery and there has been no recurrence of the lesions after 1 to 24 years of clinical follow-up. In summary, we are reporting 5 rare cases of thyrolipoma and thyrolipomatosis with distinct histologic patterns. Previously reported cases of thyrolipomatosis were reviewed and analyzed with the current cases.     

Grafting of Chitosan with Styrene and Maleic Anhydride via Nitroxide‐Mediated Radical Polymerization in Supercritical Carbon Dioxide

A novel synthesis route is used to produce chitosan‐graft‐poly(styrene‐maleic anhydride)‐OH‐TEMPO (CTS‐g‐PSMA‐T). A three‐step reaction scheme is proposed: 1) bromine 4‐OH‐TEMPO oxoammonium salt is synthesized. 2) Hydroxyl‐targeted groups in the CTS molecule are reacted with the synthesized salt in aqueous acid solution. A functionalization of 18.9% is achieved. 3) Graft copolymerization of styrene and maleic anhydride is done via NMRP by a unimolecular initiation system. The reaction is run in a dispersion in supercritical carbon dioxide (scCO₂) in the presence of camphorsulfonic acid (CSA) to avoid autopolymerization. A modified CTS with a graft content of 68% in weight is obtained.

     

CRAT missense variants cause abnormal carnitine acetyltransferase function in an early‐onset case of Leigh syndrome

Leigh syndrome, or subacute necrotizing encephalomyelopathy, is one of the most severe pediatric disorders of the mitochondrial energy metabolism. By performing whole‐exome sequencing in a girl affected by Leigh syndrome and her parents, we identified two heterozygous missense variants (p.Tyr110Cys and p.Val569Met) in the carnitine acetyltransferase (CRAT) gene, encoding an enzyme involved in the control of mitochondrial short‐chain acyl‐CoA concentrations. Biochemical assays revealed carnitine acetyltransferase deficiency in the proband‐derived fibroblasts. Functional analyses of recombinant‐purified CRAT proteins demonstrated that both missense variants, located in the acyl‐group binding site of the enzyme, severely impair its catalytic function toward acetyl‐CoA, and the p.Val569Met variant also toward propionyl‐CoA and octanoyl‐CoA. Although a single recessive variant in CRAT has been recently associated with neurodegeneration with brain iron accumulation (NBIA), this study reports the first kinetic analysis of naturally occurring CRAT variants and demonstrates the genetic basis of carnitine acetyltransferase deficiency in a case of mitochondrial encephalopathy.     

Morphometric assessment of the impact of serum thymulin immunoneutralization on pituitary cell populations in peripubertal mice

Thymulin is a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymus-pituitary communication, particularly during early life. The aim of the present study was to evaluate the impact of serum thymulin suppression from birth to peripuberty on the morphology of different pituitary cell populations in prepubertal C57Bl/6 mice. Animals were submitted to immunoneutralization of circulating thymulin from postnatal day 1 to the end of the study (age 32 days). From their 1st day of life, the animals were submitted to a protocol of intraperitoneal injections of rabbit anti-thymulin serum (alpha-FTS) and normal rabbit serum (NRS) in the controls. On their 33rd day of life, the animals were killed and their pituitaries were immediately dissected, fixed and immunostained using the EnVision system with primary antibodies against growth hormone, thyrotropin, corticotropin, gonadotropins and prolactin. Morphometry was performed by means of an image analysis system. The following parameters were calculated: volume density = Sigma cell area/reference area (RA); cell density (CD) = number of cells/RA, and cell size (expressed in microm2). Serum thymulin was measured by a rosette bioassay while pituitary hormones were assayed by radioimmunoassay. Serum prolactin, luteinizing hormone, follicle-stimulating hormone, growth hormone and thyroid-stimulating hormone were significantly lower in the alpha-FTS animals of either sex compared with the corresponding NRS counterparts. The somatotrope, lactotrope and corticotrope populations showed a significant decrease in CD, while cell hypertrophy was observed in some of the pituitary cell populations of the alpha-FTS group compared to the NRS group. In the alpha-FTS group, there were sex differences in the morphometric changes observed. Our results suggest that serum thymulin plays a significant role during early life in the postnatal maturation of endocrine cells of the mouse anterior pituitary gland.     

Evidence for linkage of nonsyndromic cleft lip with or without cleft palate to a region on chromosome 2

Results from a genome-wide screen of 10 multiplex families ascertained through probands with nonsyndromic cleft lip with or without cleft palate (CL/P) in Mexico, Argentina, and the United States yielded suggestive evidence of linkage to chromosomes 2, 6, 17 and 18. Fine mapping excluded all regions except chromosome 2. Subsequent analysis was performed on the original 10 families plus an additional 16 families using 31 markers on chromosome 2. This analysis showed intriguing evidence of linkage to 2q (Zlr=2.26, empirical P-value=0.028 in a chromosome-wide analysis). Transmission disequilibrium tests also revealed evidence of linkage and disequilibrium for two markers in this region (D2S168 and D2S1400 with P-values=0.022 and 0.006, respectively). A subset of these 26 families provided additional evidence for a susceptibility gene for CL/P on 2q, suggesting that further studies of genes in this region are warranted.     

Optimal number of implants for complete-arch implant-supported prostheses with a follow-up of at least 5 years: A systematic review and meta-analysis

Statement of problem

Consensus is lacking regarding the optimal number of implants for supporting complete-arch prostheses with good survival rates and lower prosthetic complications and marginal bone loss.