BACKGROUND:Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of chronic kidney disease in the first three decades of life. Variants in four Forkhead box (FOX) transcription factors have been associated with CAKUT. We hypothesized that other FOX genes, if highly expressed in developing kidneys, may also represent monogenic causes of CAKUT. METHODS:We here performed whole-exome sequencing (WES) in 541 families with CAKUT and generated four lists of CAKUT candidate genes: (A) 36 FOX genes showing high expression during renal development, (B) 4 FOX genes known to cause CAKUT to validate list A, (C) 80 genes that we identified as unique potential novel CAKUT candidate genes when performing WES in 541 CAKUT families and (D) 175 genes identified from WES as multiple potential novel CAKUT candidate genes. RESULTS:To prioritize potential novel CAKUT candidates in the FOX gene family, we overlapped 36 FOX genes (list A) with lists C and D of WES-derived CAKUT candidates. Intersection with list C identified a de novo FOXL2 in-frame deletion in a patient with eyelid abnormalities and ureteropelvic junction obstruction, and a homozygous FOXA2 missense variant in a patient with horseshoe kidney. Intersection with list D identified a heterozygous FOXA3 missense variant in a CAKUT family with multiple affected individuals. CONCLUSIONS:We hereby identified FOXL2, FOXA2 and FOXA3 as novel monogenic candidate genes of CAKUT, supporting the utility of a paralog-based approach to discover mutated genes associated with human disease.

CMOS (Complementary Metal Oxide Semiconductor) transmitter and receiver circuits for rotational spectroscopy are demonstrated. The IC's implemented in 65-nm CMOS consist of a 208-252 GHz transmitter and a 225-280 GHz receiver. Use of CMOS electronics can reduce the cost of electronics for rotational spectrometer application from over $50k to less than $1k. The receiver (RX) includes an on-chip antenna for air-to-chip interface, a 2nd order sub-harmonic down-conversion mixer, a low noise IF amplifier and an amplitude detector. The transmitter (TX) includes an on-chip antenna for chip-to-air interface, Fractional-N synthesizer with a frequency step less than 1 kHz with a built-in frequency shift keying circuit as well as a frequency up-converter to generate the signal at the RF. The integrated circuits were assembled into a rotational spectrometer and utilized to detect numerous gases including Ethanol and Acetone in human breath. It is the first ever demonstration of spectroscopy on pure gases as well as breath using CMOS circuits, and this work paves the way toward a more compact, affordable and efficient rotational spectroscopy system.

Forkhead transcription factor C2 (FOXC2) is a transcription factor regulating vascular and lymphatic development, and its mutations are linked to lymphedema-distichiasis syndrome. FOXC2 is also a crucial regulator of the epithelial-mesenchymal transition processes essential for tumor metastasis. Here, we report the crystal structure of the FOXC2-DNA-binding domain in complex with its cognate DNA. The crystal structure provides the basis of DNA sequence recognition by FOXC2 for the T/CAAAC motif. Helix 3 makes the majority of the DNA-protein interactions and confers the DNA sequence specificity. The computational energy calculation results also validate the structural observations. The FOXC2 and DNA complex structure provides a detailed picture of protein and DNA interactions, which allows us to predict its DNA recognition specificity and impaired functions in mutants identified in human patients.

CMOS IC technology has become an affordable means for implementing capable systems operating at 300 GHz and above. CMOS circuits have been used to generate a signal up to 1.3 THz to detect both amplitude and phase of signals up to 1.2 THz, and to detect a signal amplitude up to ~10 THz. Additionally, a transmitter and a receiver operating up to ~300 GHz for electronic smelling using rotational spectroscopy, a 30-Gb/s 300-GHz QPSK transmitter for data communication with -6-dBm output power, and an 820 GHz imaging array fabricated in CMOS have been reported. These results, along with the CMOS circuit performance in the literature, and link analyses suggest that the electronics necessary for everyday life applications in the terahertz band can be affordably realized.

The cover feature picture shows MC-1-F2, the first small-molecule inhibitor of forkhead box protein C2 (FOXC2). FOXC2 is known to be necessary for initiating and maintaining the epithelial–mesenchymal transition (EMT), a critical process of cancer metastasis. A bicycle with square wheels illustrates the cancer cells whose metastatic move was halted due to the reversal of EMT by MC-1-F2. More information can be found in the communication by J. Lee et al. on page 1359 in Issue 13, 2018 (DOI: 10.1002/cbic.201800022).

The epithelial-mesenchymal transition (EMT) has been suggested as a new target for therapeutic intervention of metastatic cancer. Forkhead box proteinC2 (FOXC2) is known to be necessary for initiating and maintaining EMT, and therefore bestows on cancer cells metastatic and cancer stem cell (CSC)-like phenotypes, allowing cells to acquire higher motility, invasiveness, self-renewal, and therapy resistance. Here, we describe the first inhibitor of FOXC2, MC-1-F2. MC-1-F2 was able to induce cadherin switching and reverse EMT through the degradation of FOXC2 and blocking of its nuclear localization. In addition, MC-1-F2 was very effective in inhibiting cancer cell migration and invasion. As the first small-molecule inhibitor of FOXC2 and the first compound targeting EMT-associated transcription factor, MC-1-F2 will pave the way for a new anticancer therapeutic agent targeting metastatic cancer and help to elucidate the network of EMT signaling pathways.

CMOS (Complementary Metal Oxide Silicon) integrated circuits (IC’s) technology is emerging as a means for realization of capable and affordable systems that operate at 300GHz and higher. Despite the fact that the unity maximum available gain frequency, fmax of NMOS transistors has peaked at ~320GHz somewhere between 65 and 32-nm technology nodes, signal generation up to 1.3THz and coherent detection up to 410GHz and incoherent detection up to ~10THz have been demonstrated using CMOS integrated circuits. Furthermore, a highly integrated rotational spectroscopy transceiver operating up to near 300GHz and imaging array operating at 820GHz have been demonstrated in CMOS. The nonlinear devices and circuit techniques that enable the operation at these frequencies beyond fmax will be described. Lastly, emerging applications, electronic nose/smelling using rotational spectroscopy that can detect and quantify concentrations of a wide variety of gases, imaging that can enable operation of autonomous systems in a wide range of weather conditions (rain, dust, snow, fog .. ), high-bandwidth communication over 1-2m long dielectric waveguides that can rival the bandwidth of optical communication systems, and electronic detectors that can make thermal imaging/night vision affordable will be discussed.

Forkhead C2 (FOXC2) transcription factor, normally regulating vascular endothelial development processes, exhibits abnormally high expression in cancer stem cells, a major culprit for cancer recurrence.It is also associated with endothelial to mesenchymal transition, a crucial process in cancer metastasis.Cancer recurrence, metastasis and angiogenesis are key elements of late stage cancers for which only very limited treatment options are available.Thus, FOXC2 can serve as an ideal target for the new generations of cancer therapeutics.We identified several candidates of FOXC2 inhibitors from helix-mimetics libraries.To understand detailed mechanisms of functional interference by the inhibitors and to optimize the inhibitors through structure-guided design approaches, we initiated structural studies of the human FOXC2 and the inhibitor complexes using X-ray crystallography methods.Crystals of FOXC2 protein in complex with the target DNA and an inhibitor have been obtained, and the crystallization conditions are being optimized.The structure analysis of the FOXC2 will facilitate understanding of its functions and provide basis for the therapeutics development targeting this protein.

We report on our progress in utilizing THz breath sensing in several bio-medical diagnostic applications. Our work bears promise in applying this technology to non-invasive analysis of blood glucose based on chemical composition of breath, as well as assessment of asthma related airway inflammation. Our most recent testing of CMOS based THz breath sensor, in the evolution of this technology towards compact and affordable implementations, is discussed.

Ewing's Sarcoma transcript-2 (EAT2) also known as SH2D1B is involved in regulation of signalling lymphocytic activation molecule (SLAM) family receptor functions. Cytoplasmic tails of SLAM family receptors contain tyrosine residues which mediate the downstream signal transduction through their phosphorylation. EAT2, composed of a single SH2 domain and a short C-terminal tail, binds to the phosphotyrosine residues and regulates SLAM family receptor signalling. We have determined the crystal structure of the human EAT2 protein in an unliganded form. Compared with the mouse EAT2-peptide complex structure, we observe conformational differences in the loops involved in ligand binding. When compared with SAP, the other single SH2 domain protein in human, EAT2 shows similar binding energies to unphosphorylated ligands. This is inconsistent to the previous data showing low affinity of EAT2 toward unphosphorylated peptides compared to SAP which shows high affinity. Additional factors other than the SH2 domains may contribute to the reported differences.

Recent advances of CMOS technology and circuits have made it an alternative for realizing capable and affordable THz systems. With process and circuit optimization, it should be possible to generate useful power and coherently detect signals at frequencies beyond 1THz, and incoherently detect signals at 40THz in CMOS.

High levels of circulating immunoglobulin G (IgG) and serum albumin (SA) are maintained through recycling by the neonatal Fc receptor (FcRn). FcRn interacts with IgG and SA in a pH-dependent manner and rescues them from lysosomal degradation. We have determined the crystal structure of extracellular domain of human FcRn, a heterodimeric complex of alpha-chain and beta 2-microglobulin, at pH 4.5. The structure was compared with the previously reported unliganded human FcRn structure at pH 8.5 and complex structures of FcRn bound to SA and/or Fc determined at acidic pHs. Structural differences are more pronounced between the two unliganded FcRn structures at pH 4.5 and pH 8.5 than between unliganded FcRn and the complex structures at acidic pHs. At acidic pH, protonation of H166 induces interactions with E54 and Y60 stabilizing the "WW loop" important for SA binding, and H161 interacts with E165 causing conformational changes of helix 3. These structural changes make the FcRn amenable for binding with SA at acidic pH. The Fc binding surface does not show any major main chain differences between the unliganded structures at pH 8.5 and pH 4.5. Side chain changes upon Fc binding were observed when compared with the complex structures. This suggests that major structural differences observed between unliganded and ligand bound structures are primarily due to pH changes rather than ligand binding.

Heart development in mammalian systems is controlled by combinatorial interactions of master cardiac transcription factors such as TBX5 and NKX2.5. They bind to promoters/enhancers of downstream targets as homo- or heteromultimeric complexes. They physically interact and synergistically regulate their target genes. To elucidate the molecular basis of the intermolecular interactions, a heterodimer and a homodimer of NKX2.5 and TBX5 were studied using X-ray crystallography. Here we report a crystal structure of human NKX2.5 and TBX5 DNA binding domains in a complex with a 19 bp target DNA and a crystal structure of TBX5 homodimer. The ternary complex structure of NKX2.5 and TBX5 with the target DNA shows physical interactions between the two proteins through Lys158 (NKX2.5), Asp140 (TBX5), and Pro142 (TBX5), residues that are highly conserved in TBX and NKX families across species. Extensive homodimeric interactions were observed between the TBX5 proteins in both crystal structures. In particular, in the crystal structure of TBX5 protein that includes the N-terminal and DNA binding domains, intermolecular interactions were mediated by the N-terminal domain of the protein. The N-terminal domain of TBX5 was predicted to be "intrinsically unstructured", and in one of the two molecules in an asymmetric unit, the N-terminal domain assumes a β-strand conformation bridging two β-sheets from the two molecules. The structures reported here may represent general mechanisms for combinatorial interactions among transcription factors regulating developmental processes.

Growing numbers of protein and nucleic acid complex structures are being determined and deposited in the Protein Data Bank and the Nucleic Acid Database. With the increasing complexity of these structures, it is challenging to analyse and visualize the three-dimensional interactions. The currently available programs for such analysis and visualization are limited in their applications. They can only analyse a subset of protein-nucleic acid complexes and require multiple iterations before obtaining plots that are suitable for presentation. An interactive web-based program, NuProPlot (http://www.nuproplot.com), has been developed which can automatically identify hydrogen, electrostatic and van der Waals interactions between proteins and nucleic acids and generate a plot showing all of the interactions. Protein-DNA and protein-RNA interactions can be visualized in simple two-dimensional schematics. Interactive schematic drawing options allow selection of the plotted area and repositioning of the individual interactions for better legibility. NuProPlot is a fully automated and user-friendly program providing various custom options. NuProPlot represents a greatly improved option for analysis and presentation of protein-nucleic acid interactions.

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

Heart development depends on timely expression of genes regulated by combinatorial interactions of master cardiac transcription factors. To elucidate the molecular basis of their interactions, a ternary complex of cardiac transcription factors, NKX2.5 and TBX5, and their target DNA was studied using X-ray crystallography. Here, the purification, crystallization and preliminary X-ray crystallographic analyses of the NKX2.5 homeodomain and TBX5 DNA-binding domain complex with a DNA element from the −252 promoter region of the atrial natriuretic factor are reported. The crystal diffracted to 2.88 Å resolution and belonged to space groupP21, with unit-cell parametersa= 69.30,b= 77.78,c= 77.60 Å, β = 108.31°. Two sets of ternary complexes are present in an asymmetric unit with a solvent content of 54%.

ABSTRACT The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors.

The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded β-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.