Analysis of genomic distributions of SARS-CoV-2 reveals a dominant strain type with strong allelic associations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID 19, continues to evolve since its first emergence in December 2019. Using the complete sequences of 1,932 SARS-CoV-2 genomes, various clustering analyses consistently identified six types of the strains. Independent of the dendrogram construction, 13 signature variations in the form of single nucleotide variations (SNVs) in protein coding regions and one SNV in the 5' untranslated region (UTR) were identified and provided a direct interpretation for the six types (types I to VI). The six types of the strains and their underlying signature SNVs were validated in two subsequent analyses of 6,228 and 38,248 SARS-CoV-2 genomes which became available later. To date, type VI, characterized by the four signature SNVs C241T (5'UTR), C3037T (nsp3 F924F), C14408T (nsp12 P4715L), and A23403G (Spike D614G), with strong allelic associations, has become the dominant type. Since C241T is in the 5' UTR with uncertain significance and the characteristics can be captured by the other three strongly associated SNVs, we focus on the other three. The increasing frequency of the type VI haplotype 3037T-14408T-23403G in the majority of the submitted samples in various countries suggests a possible fitness gain conferred by the type VI signature SNVs. The fact that strains missing one or two of these signature SNVs fail to persist implies possible interactions among these SNVs. Later SNVs such as G28881A, G28882A, and G28883C have emerged with strong allelic associations, forming new subtypes. This study suggests that SNVs may become an important consideration in SARS-CoV-2 classification and surveillance.

OPATs: Omnibus P-value association tests.

Combining statistical significances (P-values) from a set of single-locus association tests in genome-wide association studies is a proof-of-principle method for identifying disease-associated genomic segments, functional genes and biological pathways. We review P-value combinations for genome-wide association studies and introduce an integrated analysis tool, Omnibus P-value Association Tests (OPATs), which provides popular analysis methods of P-value combinations. The software OPATs programmed in R and R graphical user interface features a user-friendly interface. In addition to analysis modules for data quality control and single-locus association tests, OPATs provides three types of set-based association test: window-, gene- and biopathway-based association tests. P-value combinations with or without threshold and rank truncation are provided. The significance of a set-based association test is evaluated by using resampling procedures. Performance of the set-based association tests in OPATs has been evaluated by simulation studies and real data analyses. These set-based association tests help boost the statistical power, alleviate the multiple-testing problem, reduce the impact of genetic heterogeneity, increase the replication efficiency of association tests and facilitate the interpretation of association signals by streamlining the testing procedures and integrating the genetic effects of multiple variants in genomic regions of biological relevance. In summary, P-value combinations facilitate the identification of marker sets associated with disease susceptibility and uncover missing heritability in association studies, thereby establishing a foundation for the genetic dissection of complex diseases and traits. OPATs provides an easy-to-use and statistically powerful analysis tool for P-value combinations. OPATs, examples, and user guide can be downloaded from http://www.stat.sinica.edu.tw/hsinchou/genetics/association/OPATs.htm.

Analytical determination of the complex refractive index and the incident angle of an optically isotropic substrate by ellipsometric parameters and reflectance.

An analytical solution for the determination of both angle of incidence (AOI) and the complex refractive index from combined ellipsometric and reflectometric measurements at optically isotropic substrates is presented. Conventional ellipsometers usually measure flat surfaces because the curvatures of the surface alter the reflected or transmitted light, which causes experimental errors due to the deviation of the incident angle. However, in real industrial applications, the shapes of samples are usually curved or even free-form. In this case, the knowledge of the AOI is essential. The proposed method provides a simple way to measure the AOI and the complex refractive index of nonplanar samples without extra or complicated hardware.

A Novel Spo11 Homologue Functions as a Positive Regulator in Cyst Differentiation in Giardia lamblia.

Giardia lamblia persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during Giardia encystation, suggesting parasexual recombination processes of this protozoan. Giardia possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the cwp promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate cwp gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of cwp1-3 and myb2 genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Arabidopsis Spo11 reduced the levels of cwp1-3 and myb2 gene expression and cyst formation. Targeted disruption of spo11 gene with CRISPR/Cas9 system led to a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for Giardia differentiation into cyst.

Enhanced Photocarrier Generation with Selectable Wavelengths by M-Decorated-CuInS2 Nanocrystals (M = Au and Pt) Synthesized in a Single Surfactant Process on MoS2 Bilayers.

A facile approach for the synthesis of Au- and Pt-decorated CuInS2 nanocrystals (CIS NCs) as sensitizer materials on the top of MoS2 bilayers is demonstrated. A single surfactant (oleylamine) is used to prepare such heterostructured noble metal decorated CIS NCs from the pristine CIS. Such a feasible way to synthesize heterostructured noble metal decorated CIS NCs from the single surfactant can stimulate the development of the functionalized heterostructured NCs in large scale for practical applications such as solar cells and photodetectors. Photodetectors based on MoS2 bilayers with the synthesized nanocrystals display enhanced photocurrent, almost 20-40 times higher responsivity and the On/Off ratio is enlarged one order of magnitude compared with the pristine MoS2 bilayers-based photodetectors. Remarkably, by using Pt- or Au-decorated CIS NCs, the photocurrent enhancement of MoS2 photodetectors can be tuned between blue (405 nm) to green (532 nm). The strategy described here acts as a perspective to significantly improve the performance of MoS2 -based photodetectors with the controllable absorption wavelengths in the visible light range, showing the feasibility of the possible color detection.

Actin cytoskeleton remodeling drives epithelial-mesenchymal transition for hepatoma invasion and metastasis in mice.

High invasiveness is a hallmark of human hepatocellular carcinoma (HCC). Large tumors predict invasion and metastasis. Epithelial-mesenchymal transition (EMT) is crucial for cancer invasion and metastasis. However, the mechanisms whereby large tumors tend to undergo EMT remain unclear. We conducted a subgenome-wide screen and identified KLHL23 as an HCC invasion suppressor by inhibiting EMT. KLHL23 binds to actin and suppresses actin polymerization. KLHL23 silencing induced filopodium and lamellipodium formation. Moreover, EMT was suppressed by KLHL23 through its action on actin dynamics. Traditionally, actin cytoskeleton remodeling is downstream of EMT reprogramming. It is therefore intriguing to ask why and how KLHL23 inversely regulates EMT. Activation of actin cytoskeleton remodeling by either KLHL23 silencing or treatment with actin cytoskeleton modulators augmented cellular hypoxic responses in a cell-density-dependent manner, resulting in hypoxia-inducible factor (HIF) and Notch signals and subsequent EMT. Environmental hypoxia did not induce EMT unless actin cytoskeleton remodeling was simultaneously activated and only when cells were at high density. The resulting EMT was reversed by either adenosine 5'-triphosphate supplementation or actin polymerization inhibitors. Down-regulation of KLHL23 was associated with invasion, metastasis, and poor prognosis of HCC and pancreatic cancer. Correlations of tumor size with EMT and inverse association of expression of KLHL23 with HIF/Notch signals were further validated in patient-derived xenograft HCCs in mice. CONCLUSION: Simultaneously activation of actin cytoskeleton remodeling by intrinsic (such as KLHL23 down-regulation) or microenvironment cues is crucial for cell-density-dependent and hypoxia-mediated EMT, providing a mechanistic link between large tumor size and invasion/metastasis. Our findings provide a means of developing the prevention and treatment strategies for tumor invasion and metastasis. (Hepatology 2018;67:2226-2243).

Immunomodulatory Effects of Current Targeted Therapies on Hepatocellular Carcinoma: Implication for the Future of Immunotherapy.

Multikinase inhibitors with antiangiogenic properties used to be standard therapy for patients with advanced hepatocellular carcinoma (HCC). Recently, several antiangiogenic agents (lenvatinib, cabozantinib, and ramucirumab) have demonstrated antitumor activity for advanced HCC in randomized controlled trials. However, the landscape of drug development for HCC may change dramatically with the advent of immune checkpoint inhibitor therapy, particularly the anti-programmed cell death-1 (anti-PD1) agents. In addition, early-phase clinical trials of combination of anti-PD-1 and antiangiogenic agents have shown very promising anti-tumor activity in patients with advanced HCC. Therefore, the critical research questions at present are whether this combination strategy will be the next generation of standard therapy and which antiangiogenic agents will be the optimal partner for the combination. All of the 4 multikinase inhibitors for HCC (sorafenib, regorafenib, lenvatinib, and cabozantinib) have been reported to have immune modulatory effects. The authors systematically reviewed the pre-clinical evidence of their immune modulatory effects to explore whether these effects were mediated by angiogenesis inhibition or by other "off-target" effects on the tumor microenvironment. Studies of sorafenib comprised the majority (58 of the 71) of the research articles reviewed. Potentially beneficial effects on anti-tumor immunity may result from increased M1 polarization of macrophages and stimulation of CD8 T cell function. On the other hand, high dosage of the kinase inhibitors in pre-clinical models and hypoxia associated with angiogenesis may contribute to immune suppression in the tumor microenvironment. Sorafenib and other multikinase inhibitors may promote anti-tumor immunity through modulation of multiple immune cell types as well as the tumor microenvironment. The optimal immune modulatory dosage should be defined to facilitate design of future combination regimens.

Phase-Engineered PtSe2 -Layered Films by a Plasma-Assisted Selenization Process toward All PtSe2 -Based Field Effect Transistor to Highly Sensitive, Flexible, and Wide-Spectrum Photoresponse Photodetectors.

The formation of PtSe2 -layered films is reported in a large area by the direct plasma-assisted selenization of Pt films at a low temperature, where temperatures, as low as 100 °C at the applied plasma power of 400 W can be achieved. As the thickness of the Pt film exceeds 5 nm, the PtSe2 -layered film (five monolayers) exhibits a metallic behavior. A clear p-type semiconducting behavior of the PtSe2 -layered film (≈trilayers) is observed with the average field effective mobility of 0.7 cm2 V-1 s-1 from back-gated transistor measurements as the thickness of the Pt film reaches below 2.5 nm. A full PtSe2 field effect transistor is demonstrated where the thinner PtSe2 , exhibiting a semiconducting behavior, is used as the channel material, and the thicker PtSe2 , exhibiting a metallic behavior, is used as an electrode, yielding an ohmic contact. Furthermore, photodetectors using a few PtSe2 -layered films as an adsorption layer synthesized at the low temperature on a flexible substrate exhibit a wide range of absorption and photoresponse with the highest photocurrent of 9 µA under the laser wavelength of 408 nm. In addition, the device can maintain a high photoresponse under a large bending stress and 1000 bending cycles.

Phase-Engineered Type-II Multimetal-Selenide Heterostructures toward Low-Power Consumption, Flexible, Transparent, and Wide-Spectrum Photoresponse Photodetectors.

Phase-engineered type-II metal-selenide heterostructures are demonstrated by directly selenizing indium-tin oxide to form multimetal selenides in a single step. The utilization of a plasma system to assist the selenization facilitates a low-temperature process, which results in large-area films with high uniformity. Compared to single-metal-selenide-based photodetectors, the multimetal-selenide photodetectors exhibit obviously improved performance, which can be attributed to the Schottky contact at the interface for tuning the carrier transport, as well as the type-II heterostructure that is beneficial for the separation of the electron-hole pairs. The multimetal-selenide photodetectors exhibit a response to light over a broad spectrum from UV to visible light with a high responsivity of 0.8 A W-1 and an on/off current ratio of up to 102 . Interestingly, all-transparent photodetectors are successfully produced in this work. Moreover, the possibility of fabricating devices on flexible substrates is also demonstrated with sustainable performance, high strain tolerance, and high durability during bending tests.

Man with severe abdominal pain.

CLINICAL INTRODUCTION: A 59-year-old man with no medical history presented to the ED with abdominal distension, vomiting and diffuse abdominal pain after drinking seven cups of white gourd drink (an authentic Asian drink composed mainly of white gourd). A chest radiograph was performed (figure 1).emermed;35/9/571/F1F1F1Figure 1Chest radiograph. AP, anteroposterior. QUESTION: What is the most likely diagnosis?Crescent in doughnut sign, intussusceptionFalciform ligament sign, pneumoperitoneumStepladder sign, small bowel obstructionCoffee-bean sign, sigmoid colon volvulus.

Efficient demodulation scheme for rolling-shutter-patterning of CMOS image sensor based visible light communications.

Recently even the low-end mobile-phones are equipped with a high-resolution complementary-metal-oxide-semiconductor (CMOS) image sensor. This motivates using a CMOS image sensor for visible light communication (VLC). Here we propose and demonstrate an efficient demodulation scheme to synchronize and demodulate the rolling shutter pattern in image sensor based VLC. The implementation algorithm is discussed. The bit-error-rate (BER) performance and processing latency are evaluated and compared with other thresholding schemes.

CuI/Oxalic Diamide-Catalyzed Cross-Coupling of Thiols with Aryl Bromides and Chlorides.

We report a general copper-catalyzed cross-coupling of thiols with aryl halides by using N-aryl-N'-alkyl oxalic diamide (L3) or N,N'-dialkyl oxalic diamide (L5) as the ligand. Both aryl and alkyl thiols can be coupled with unactivated aryl bromides and chlorides to give the desired products in good yields. Furthermore, this system features a broad substrate scope and good tolerance of functional groups. Importantly, the oxalic diamides are stable and can be prepared easily from commercially available and cheap starting materials.

Transfer-Free Growth of Atomically Thin Transition Metal Disulfides Using a Solution Precursor by a Laser Irradiation Process and Their Application in Low-Power Photodetectors.

Although chemical vapor deposition is the most common method to synthesize transition metal dichalcogenides (TMDs), several obstacles, such as the high annealing temperature restricting the substrates used in the process and the required transfer causing the formation of wrinkles and defects, must be resolved. Here, we present a novel method to grow patternable two-dimensional (2D) transition metal disulfides (MS2) directly underneath a protective coating layer by spin-coating a liquid chalcogen precursor onto the transition metal oxide layer, followed by a laser irradiation annealing process. Two metal sulfides, molybdenum disulfide (MoS2) and tungsten disulfide (WS2), are investigated in this work. Material characterization reveals the diffusion of sulfur into the oxide layer prior to the formation of the MS2. By controlling the sulfur diffusion, we are able to synthesize continuous MS2 layers beneath the top oxide layer, creating a protective coating layer for the newly formed TMD. Air-stable and low-power photosensing devices fabricated on the synthesized 2D WS2 without the need for a further transfer process demonstrate the potential applicability of TMDs generated via a laser irradiation process.

SMART: Statistical Metabolomics Analysis-An R Tool.

Metabolomics data provide unprecedented opportunities to decipher metabolic mechanisms by analyzing hundreds to thousands of metabolites. Data quality concerns and complex batch effects in metabolomics must be appropriately addressed through statistical analysis. This study developed an integrated analysis tool for metabolomics studies to streamline the complete analysis flow from initial data preprocessing to downstream association analysis. We developed Statistical Metabolomics Analysis-An R Tool (SMART), which can analyze input files with different formats, visually represent various types of data features, implement peak alignment and annotation, conduct quality control for samples and peaks, explore batch effects, and perform association analysis. A pharmacometabolomics study of antihypertensive medication was conducted and data were analyzed using SMART. Neuromedin N was identified as a metabolite significantly associated with angiotensin-converting-enzyme inhibitors in our metabolome-wide association analysis (p = 1.56 × 10(-4) in an analysis of covariance (ANCOVA) with an adjustment for unknown latent groups and p = 1.02 × 10(-4) in an ANCOVA with an adjustment for hidden substructures). This endogenous neuropeptide is highly related to neurotensin and neuromedin U, which are involved in blood pressure regulation and smooth muscle contraction. The SMART software, a user guide, and example data can be downloaded from http://www.stat.sinica.edu.tw/hsinchou/metabolomics/SMART.htm .

Fabrication of Multilayer Borophene on Insulator Structure.

The X-ray photoelectron spectroscopy spectra indicate the peak of BB bonds, implying that the elemental boron structure might be formed after the process. The multilayer ß-borophene is directly observed by transmission electron microscopy (TEM) and the lattice parameters are valid. The middle SiNx layer also can be identified in TEM image. Furthermore, the 1.61 eV bandgap of the multilayer ß-borophene is announced in this study.

Comparing the Sensitivity, Specificity, and Predictive Values of the Montreal Cognitive Assessment and Mini-Mental State Examination When Screening People for Mild Cognitive Impairment and Dementia in Chinese Population.

BACKGROUND: The study compared the sensitivity, specificity, and diagnostic value of the Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) in screening for mild cognitive impairment (MCI) and dementia. METHODS: A cross-sectional descriptive design was used, and 142 participants were screened for MCI and mild dementia by using the MoCA and MMSE. The receiver operating characteristic curves and the cutoff scores with the largest area under the curve (AUC) were determined and compared to calculate the sensitivity, specificity, and diagnostic value (positive predictive value [PPV] and negative predictive value [NPV]). RESULTS: The optimal MoCA cutoff scores for MCI and dementia were 24 and 20, respectively. According to these scores, the sensitivities were 0.88 and 0.79, the specificities were 0.74 and 0.80, the AUCs were 0.91 and 0.87, the PPVs were 0.93 and 0.74, and the NPVs were 0.74 and 0.87, respectively. The optimal cutoff MMSE scores for MCI and dementia were 27 and 24, respectively. Hence, the sensitivities were 0.88 and 0.84, the specificities were 0.70 and 0.86, the AUCs were 0.88 and 0.89, the PPVs were 0.94 and 0.80, and the NPVs were 0.81 and 0.88, respectively. CONCLUSION: In the Chinese population, the MoCA is more efficient in screening for MCI than for dementia, whereas the MMSE is more efficient in screening for dementia than for MCI. The MoCA and MMSE can be used by clinical staffs for quick and accurate cognitive impairment screening, thus facilitating early and appropriate clinical intervention and treatment.

The reliability, validity, sensitivity, specificity and predictive values of the Chinese version of the Rowland Universal Dementia Assessment Scale.

AIMS AND OBJECTIVES: The purpose of this study was to translate the Rowland Universal Dementia Assessment Scale into Chinese and to evaluate the psychometric properties (reliability and validity) and the diagnostic properties (sensitivity, specificity and predictive values) of the Chinese version of the Rowland Universal Dementia Assessment Scale. BACKGROUND: The accurate detection of early dementia requires screening tools with favourable cross-cultural linguistic and appropriate sensitivity, specificity, and predictive values, particularly for Chinese-speaking populations. DESIGN: This was a cross-sectional, descriptive study. METHODS: Overall, 130 participants suspected to have cognitive impairment were enrolled in the study. A test-retest for determining reliability was scheduled four weeks after the initial test. Content validity was determined by five experts, whereas construct validity was established by using contrasted group technique. The participants' clinical diagnoses were used as the standard in calculating the sensitivity, specificity, positive predictive value and negative predictive value. RESULTS: The study revealed that the Chinese version of the Rowland Universal Dementia Assessment Scale exhibited a test-retest reliability of 0.90, an internal consistency reliability of 0.71, an inter-rater reliability (kappa value) of 0.88 and a content validity index of 0.97. Both the patients and healthy contrast group exhibited significant differences in their cognitive ability. The optimal cut-off points for the Chinese version of the Rowland Universal Dementia Assessment Scale in the test for mild cognitive impairment and dementia were 24 and 22, respectively; moreover, for these two conditions, the sensitivities of the scale were 0.79 and 0.76, the specificities were 0.91 and 0.81, the areas under the curve were 0.85 and 0.78, the positive predictive values were 0.99 and 0.83 and the negative predictive values were 0.96 and 0.91 respectively. CONCLUSION: The Chinese version of the Rowland Universal Dementia Assessment Scale exhibited sound reliability, validity, sensitivity, specificity and predictive values. RELEVANCE TO CLINICAL PRACTICE: This scale can help clinical staff members to quickly and accurately diagnose cognitive impairment and provide appropriate treatment as early as possible.

Applying genome-wide gene-based expression quantitative trait locus mapping to study population ancestry and pharmacogenetics.

BACKGROUND: Gene-based analysis has become popular in genomic research because of its appealing biological and statistical properties compared with those of a single-locus analysis. However, only a few, if any, studies have discussed a mapping of expression quantitative trait loci (eQTL) in a gene-based framework. Neither study has discussed ancestry-informative eQTL nor investigated their roles in pharmacogenetics by integrating single nucleotide polymorphism (SNP)-based eQTL (s-eQTL) and gene-based eQTL (g-eQTL). RESULTS: In this g-eQTL mapping study, the transcript expression levels of genes (transcript-level genes; T-genes) were correlated with the SNPs of genes (sequence-level genes; S-genes) by using a method of gene-based partial least squares (PLS). Ancestry-informative transcripts were identified using a rank-score-based multivariate association test, and ancestry-informative eQTL were identified using Fisher's exact test. Furthermore, key ancestry-predictive eQTL were selected in a flexible discriminant analysis. We analyzed SNPs and gene expression of 210 independent people of African-, Asian- and European-descent. We identified numerous cis- and trans-acting g-eQTL and s-eQTL for each population by using PLS. We observed ancestry information enriched in eQTL. Furthermore, we identified 2 ancestry-informative eQTL associated with adverse drug reactions and/or drug response. Rs1045642, located on MDR1, is an ancestry-informative eQTL (P = 2.13E-13, using Fisher's exact test) associated with adverse drug reactions to amitriptyline and nortriptyline and drug responses to morphine. Rs20455, located in KIF6, is an ancestry-informative eQTL (P = 2.76E-23, using Fisher's exact test) associated with the response to statin drugs (e.g., pravastatin and atorvastatin). The ancestry-informative eQTL of drug biotransformation genes were also observed; cross-population cis-acting expression regulators included SPG7, TAP2, SLC7A7, and CYP4F2. Finally, we also identified key ancestry-predictive eQTL and established classification models with promising training and testing accuracies in separating samples from close populations. CONCLUSIONS: In summary, we developed a gene-based PLS procedure and a SAS macro for identifying g-eQTL and s-eQTL. We established data archives of eQTL for global populations. The program and data archives are accessible at http://www.stat.sinica.edu.tw/hsinchou/genetics/eQTL/HapMapII.htm. Finally, the results from our investigations regarding the interrelationship between eQTL, ancestry information, and pharmacodynamics provide rich resources for future eQTL studies and practical applications in population genetics and medical genetics.

The effect of anions in the synthesis and structure of pyrazolylamidino copper(II) complexes.

Six new pyrazolylamidino Cu(II) complexes are synthesized directly from the reactions of Cu(X)2 salts (X = ClO4-, BF4-, or Cl-) and pyrazole (pzH) in nitrile solution (RCN, R = Me or Et) at 298 K via the metal-mediated coupling of RCN with pzH: [Cu(HNC(R)pz)2(X)2] (X = ClO4- or BF4-, R = Me, 1 or 7 and Et, 2 or 8, respectively) and dichloro Cu(II) complexes [Cu2Cl2(µ-Cl)2(HNC(Me)pz)2] (3) and [CuCl2(HNC(Et)pz)] (4). Four more new complexes, [Cu2(µ-Cl)2(HNC(Me)pz)2(pzH)2][X]2 (X = ClO4-, 5 and BF4-, 9) and [Cu2(µ-Cl)2(HNC(Et)pz)2(pzH)2(X)2] (X = ClO4-, 6 and BF4-, 10), are obtained indirectly from the anion substitution reaction with Cl- ions in 1 and 7, and 2 and 8, respectively. All complexes are characterized by EA, FTIR, UV-vis and EPR spectroscopy and X-ray crystallographic analyses. HNC(Et)pz or pzH is unobserved in both the nitrile-exchange reaction of 2 to d6-1 and the anion-substitution reaction of 2 to d6-5 in the CD3CN solution. The 1H NMR results reveal that the pzH-RCN coupling is intramolecular and reversible on a Cu(II) center. The crystal structures of these complexes show diverse supramolecular assemblies through imino NH⋯anion hydrogen bonds and pyrazolylamidino pz-pz (π⋯π) and pz-Cu(II) (π⋯metal) interactions. EPR results suggest weak magnetic couplings between Cu(II) centers in the polynuclear Cu(II) complexes. The yield and rate of the formation of 1 are higher in the reaction of Cu(ClO4)2 with a 4-fold molar excess of pzH compared with a 2-fold excess, indicating that [Cu(pzH)4]2+ is the more active species for pzH-RCN coupling. The highest rate for the formation of 1 is achieved when [Cu(pzH)4(ClO4)2] is used in MeCN solution. Thus, a plausible synthetic path for synthesizing pyrazolylamidino Cu(II) complexes is established. An intermediate species, [Cu(HNC(Me)pz)2(pzH)2][ClO4]2 (1a), is proposed for the synthetic process based on spectroscopic studies and DFT calculations. The reaction of [Cu(pzH)4X2] (X = ClO4-, Cl-, NO3-, or BF4-) in MeCN solution suggests that the lability of coordinated anions upon nitrile substitution affects the rate of the formation of bis-pyrazolylamidino Cu(II) complexes.