Exome analysis of Smith-Magenis-like syndrome cohort identifies de novo likely pathogenic variants.

Smith-Magenis syndrome (SMS), a neurodevelopmental disorder characterized by dysmorphic features, intellectual disability (ID), and sleep disturbances, results from a 17p11.2 microdeletion or a mutation in the RAI1 gene. We performed exome sequencing on 6 patients with SMS-like phenotypes but without chromosomal abnormalities or RAI1 variants. We identified pathogenic de novo variants in two cases, a nonsense variant in IQSEC2 and a missense variant in the SAND domain of DEAF1, and candidate de novo missense variants in an additional two cases. One candidate variant was located in an alpha helix of Necdin (NDN), phased to the paternally inherited allele. NDN is maternally imprinted within the 15q11.2 Prader-Willi Syndrome (PWS) region. This can help clarify NDN's role in the PWS phenotype. No definitive pathogenic gene variants were detected in the remaining SMS-like cases, but we report our findings for future comparison. This study provides information about the inheritance pattern and recurrence risk for patients with identified variants and demonstrates clinical and genetic overlap of neurodevelopmental disorders. Identification and characterization of ID-related genes that assist in development of common developmental pathways and/or gene-networks, may inform disease mechanism and treatment strategies.

Combined alpha-delta platelet storage pool deficiency is associated with mutations in GFI1B.

Combined alpha-delta platelet storage pool deficiency is characterized by the absence or reduction in the number of both alpha granules and dense bodies. This disorder can have variable severity as well as a variable inheritance pattern. We describe two patients from unrelated families with combined alpha-delta storage pool deficiency due to mutations in GFI1B, a zinc finger protein known to act as a transcriptional repressor of various genes. We demonstrate that this disease is associated with either a heterozygous mutation (de novo or familial) abrogating the binding of the zinc fingers with the promoter of its target genes, or by hypomorphic biallelic mutations in GFI1B leading to autosomal recessive inheritance.

Clinical and molecular phenotyping of a child with Hermansky-Pudlak syndrome-7, an uncommon genetic type of HPS.

PURPOSE: Hermansky-Pudlak syndrome (HPS) is a rare inherited disorder with ten reported genetic types; each type has defects in subunits of either Adaptor Protein-3 complex or Biogenesis of Lysosome-related Organelles Complex (BLOC)-1, -2, or -3. Very few patients with BLOC-1 deficiency (HPS-7, -8, and -9 types) have been diagnosed. We report results of comprehensive clinical testing and molecular analyses of primary fibroblasts from a new case of HPS-7. RESULTS: A 6-year old Paraguayan male presented with hypopigmentation, ocular albinism, nystagmus, reduced visual acuity, and easy bruising. He also experienced delayed motor and language development as a very young child; head and chest trauma resulted in intracranial hemorrhage with subsequent right hemiparesis and lung scarring. There was no clinical evidence of immunodeficiency or colitis. Whole mount transmission electron microscopy revealed absent platelet delta granules; platelet aggregation testing was abnormal. Exome sequencing revealed a homozygous nonsense mutation in the Dystrobrevin binding protein 1 (DTNBP1) gene [NM_032122.4: c.307C>T; p.Gln103*], previously reported in a Portuguese adult. The gene encodes the dysbindin subunit of BLOC-1. Dysbindin protein expression was negligible in our patient's dermal fibroblasts, while his DTNBP1 mRNA level was similar to that of a normal control. CONCLUSIONS: Comprehensive clinical evaluation of the first pediatric case reported with HPS-7 reveals oculocutaneous albinism and platelet storage pool deficiency; his phenotype is consistent with findings in other patients with BLOC-1 disorders. This patient's markedly reduced Dysbindin protein expression in HPS-7 resulted from a mechanism other than nonsense mediated decay.

Identification of a novel mutation in HPS6 in a patient with hemophilia B and oculocutaneous albinism.

PURPOSE: Hemophilia B, an X-linked disease, manifests with recurrent soft tissue bleeding episodes. Hermansky-Pudlak syndrome, a rare autosomal recessive disorder, is characterized by oculocutaneous albinism and an increased tendency to bleed due to a platelet storage pool defect. We report a novel mutation in HPS6 in a Caucasian man with hemophilia B and oculocutaneous albinism. RESULTS: The patient was diagnosed with hemophilia B at age 4months due to recurrent soft tissue bleeding episodes, and he was also diagnosed with Hermansky-Pudlak syndrome at 32years of age due to unexplained oculocutaneous albinism. His factor IX level was markedly reduced at 13%; whole exome and Sanger sequencing showed the Durham mutation in F9 (NM_000133.3). The diagnosis of Hermansky-Pudlak syndrome subtype 6 was established by demonstrating absence of platelet delta granules on whole mount electron microscopy, an abnormal secondary wave in platelet aggregation studies, and a novel homozygous c.1114 C>T (p.Arg372*) mutation in HPS6 (NM_024747.5) on exome analysis and Sanger sequencing. Clinical phenotyping revealed no evidence of recurrent or unusual infections, interstitial lung disease or pulmonary fibrosis, or neurological disorders. The patient was treated with fresh frozen plasma, recombinant factor IX, and aminocaproic acid. Treatment with desmopressin was added to his regimen after he was diagnosed with Hermansky-Pudlak syndrome. Treatment of bleeding episodes results in effective hemostasis, and the patient has not required platelet or blood product transfusions. CONCLUSIONS: This report highlights the need to consider Hermansky-Pudlak syndrome as an etiology of oculocutaneous albinism even in patients with known hematologic disorders associated with bleeding. Identification of a novel mutation in HPS6 in an individual with hemophilia B shows that, although quite rare, patients may be diagnosed with two independent inherited bleeding disorders. No evidence of lung disease was found in this adult patient with Hermansky-Pudlak syndrome subtype 6.

Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel).

Transcription factor zinc finger and BTB domain 1 is essential for lymphocyte development.

Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell development did not progress beyond double-negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild-type hematopoietic cells corrected the defect, which was traced to a deletion involving five contiguous genes at the transgene insertion site on chromosome 12C3. Complementation using bacterial artificial chromosome transgenesis implicated zinc finger BTB-POZ domain protein 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted disruption of Zbtb1 recapitulated the T(-)B(+)NK(-) SCID phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes, and pre-B cells, highlighting its essential role in lymphoid development.

Noncovalent polymerization of mesogens crystallizes lysozyme: correlation between nonamphiphilic lyotropic liquid crystal phase and protein crystal formation.

Crystallization of proteins is important for fundamental studies and biopharmaceutical development but remains largely an empirical science. Here, we report the use of organic salts that can form a class of unusual nonamphiphilic lyotropic liquid crystals to crystallize the protein lysozyme. Certain nonamphiphilic organic molecules with fused aromatic rings and two charges can assemble into stable thread-like noncovalent polymers that may further form liquid crystal phases in water, traditionally termed chromonic liquid crystals. Using five of these mesogenic molecules as additives to induce protein crystallization, we discover that molecules that can form liquid crystal phases in water are highly effective at inducing the crystal formation of lysozyme, even at concentrations significantly lower than that required for forming liquid crystal phases. This result reveals an example of inducing protein crystallization by the molecular assembly of the additives, and is consistent with a new mechanism by which the strong hydration of an assembly process provides a gradual means to compete for the water molecules to enable solvated proteins to form crystals.

Primary Hepatic Lymphoma Masquerading as Symptomatic Hypercalcemia.

Primary lymphoma of the liver is extremely rare, and is more common among immunocompromised patients. It typically occurs after the fifth decade of life and has a male predominance. It often presents with diagnostic difficulties to both clinicians and pathologists as most cases have a solitary or multiple mass lesions in the liver with normal alpha-fetoprotein levels. Chemotherapy is the standard of therapy. Here, we describe a unique case of primary hepatic lymphoma in an elderly immunocompetent female who presented with symptomatic hypercalcemia.

Nonamphiphilic assembly in water: polymorphic nature, thread structure, and thermodynamic incompatibility.

Self-assembly of large quantities of entirely water-soluble molecules is entropically challenging. In this work, we describe the design and synthesis of water-soluble aromatic (dichromonyl) molecules that can form nonamphiphilic assemblies and the so-called chromonic liquid crystal phase in water. We discover a new molecule, 5'DSCG-diviol, that exhibits a large birefringent phase, and we show that the formation of this unique class of nonamphiphilic lyotropic liquid crystal shares enormous similarity to the polymorphism observed for crystal formation. Small-angle neutron scattering (SANS) revealed a concentration-independent rod-shaped assembly at concentrations below and above the formation of liquid crystal phase. Adding a small percentage of monoanionic aromatic molecules to the liquid crystal resulted in the elimination of the liquid crystal phase, but addition of dianionic aromatic molecules retained the liquid crystal phase. Together, these results suggest a new assembly structure for nonamphiphilic molecules in water, which is comprised of long threads of small molecules connected by salt bridges stacked over aromatic groups, with the molecular threads heavily hydrated with solvent water. Furthermore, mixing molecules with different structures can result in new liquid crystalline materials, or in segregation of the molecules into different solvation volumes, each of which contains only one type of molecule. The unusual thermodynamic incompatibility of entirely water-soluble molecules also supports the model of molecular threads, in which two polymer-like assemblies do not mix.

Identifying the important structural elements of brominated furanones for inhibiting biofilm formation by Escherichia coli.

A collection of structurally closely related furanones was synthesized to identify the most important structural elements in brominated furanones for inhibiting the formation of bacterial biofilms. The results suggest that a conjugated exocyclic vinyl bromide on the furanone ring is the most important structural element for the non-toxic but inhibition activity for Escherichia coli biofilm formation. Furanones bearing monosubstituted bromide groups on saturated carbons were found to have a toxic effect that attenuates the bacterial growth.

Assessment of counselling for acute diarrhoea in German pharmacies: a simulated patient study.

OBJECTIVES: The aim of this study was to assess the quality of counselling provided for acute diarrhoea and to evaluate the role of the patient's approach and different user groups in determining the outcome of counselling. METHODS: The simulated patient methodology was used in all 21 community pharmacies in a north-eastern German city. Four different scenarios related to self-medication of acute diarrhoea were developed and used in all the pharmacies (a total of 84 visits). The assessment form, completed immediately postvisit by the simulated patient, included 9 objective items scored using dichotomous scales to produce a scale from 0 to 9. After evaluating the data, every pharmacy received individual performance feedback to encourage behavioural changes and improve the quality of the counselling provided. KEY FINDINGS: Overall, the quality of counselling was poor (mean score of 3.3/9 (37%)). The most common information provided was about dosage (87% of interactions), while the least common information given was about side effects (4% of interactions). The main effect was seen when comparing the product and symptom requests (F(1,60) = 24.748, P < 0.001, ωp2 = 0.277). There was no effect resulting from different user groups (F(1,28) = 0.237, P = 0.630, ωp2 = -0.026) and no interaction between the type of request and different user groups (F(1,28) = 3.395, P = 0.076, ωp2 = 0.073). CONCLUSIONS: This study highlighted the current deficits in appropriate counselling provided by community pharmacies in Germany.

Saddle Pulmonary Embolism with Thrombus in Transit across a Patent Foramen Ovale.

This is the case of a 25-year-old obese man who presented with acute shortness of breath, chest pain, and palpitations. Of note, he lives a sedentary lifestyle and was recently hospitalized for incision and drainage of a left foot abscess. On presentation he was tachypnoeic, tachycardiac, and hypoxic but blood pressure was stable. Laboratory studies were significant for elevated D-dimer and mildly increased troponin. On further investigation he was found to have a saddle pulmonary embolism with massive clot burden. Echocardiogram revealed thrombus in transit and McConnell's sign. He underwent surgical embolectomy and closure of a patent foramen ovale. This is a particularly rare case, especially in such a young patient. Because this is a rare diagnosis, with insufficient data, there is no formally established treatment guideline. However, in patients who are good surgical candidates, studies have shown better outcome with surgical embolectomy as compared to anticoagulation alone or thrombolysis.

Colorectal cancer development and advances in screening.

Most colon tumors develop via a multistep process involving a series of histological, morphological, and genetic changes that accumulate over time. This has allowed for screening and detection of early-stage precancerous polyps before they become cancerous in individuals at average risk for colorectal cancer (CRC), which may lead to substantial decreases in the incidence of CRC. Despite the known benefits of early screening, CRC remains the second leading cause of cancer-related deaths in the United States. Hence, it is important for health care providers to have an understanding of the risk factors for CRC and various stages of disease development in order to recommend appropriate screening strategies. This article provides an overview of the histological/molecular changes that characterize the development of CRC. It describes the available CRC screening methods and their advantages and limitations and highlights the stages of CRC development in which each screening method is most effective.

[Using the simulated patient methodology to assess counselling for acute diarrhoea - evidence from Germany]. / Die Beratungsqualität von Apotheken bei akutem Durchfall - Eine Analyse unter Verwendung der "Simulated Patient"-Methode.

OBJECTIVES: First, to assess the quality of counselling for acute diarrhoea; second, to evaluate the patient's approach and different user groups as a determinant of counselling. METHODS: The simulated patient methodology was used in all 21 community pharmacies in a north-eastern German city with a population of about 63,000. Four scenarios related to self-medication for acute diarrhoea were developed and used in all pharmacies (total: 84 visits). Two scenarios were direct product-based requests for loperamide (scenario 1: a 74-year old woman with diabetes and hypertension; scenario 3: a 30-year old man with no primary disease). Scenario 2 and 4 were symptom-based requests asking for medicine for acute diarrhoea (scenario 2: a 74-year old woman with diabetes and hypertension; scenario 4: a 30-year old man with no primary disease). The assessment sheet included 9 objective items relating to the pharmacological advice to avoid a subjective evaluation by the mystery shoppers (e. g., the friendliness of the customer contact). Simulated patient visits were conducted covertly by five untrained female master students. After evaluation of the data every pharmacy received an individual performance feedback to encourage behavioural change and improve counselling quality. RESULTS: Overall, the quality of counselling was quite poor (277 out of 756 possible points). The most commonly provided information was dosage (86.9 %); information on adverse effects was least commonly provided (3.6 %). Furthermore, there was a huge difference in the counselling quality between the pharmacies (minimum 4 points, maximum 20 points out of 36 possible points). The symptom-based requests scored significantly better (95 and 85 out of 189 possible points) than the direct product-based requests (42 and 55 out of 189 possible points). The symptom-based requests had a significantly better counselling quality for an older woman with primary disease than for a younger man without any primary disease. This difference was not observed with the direct product-based requests.

Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system.

This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation.

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen.

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.