Association of DAZ and DAZLA Gene Loci with Spermatogenesis in Patients with Idiopathic Azoospermia and Severe Oligospermia.

Aim: To explore the relationship between DAZ (Deleted in Azoospermia, DAZ) and DAZLA (Deleted in Azoospermia-like autosomal) gene deletion and male idiopathic azoospermia and oligozoospermia. Methods: 80 patients with azoospermia (azoospermia group) and 80 patients with oligozoospermia (oligozoospermia group) who were treated at our hospital from April 2021 to April 2023, and male volunteers who underwent health examinations at our hospital during the same period were selected as the control group, The incidence of DAZ and DAZLA gene locus deletion in three groups of men was detected by polymerase chain reaction (PCR), and the differences of reproductive hormone levels and main semen parameters among the three groups were compared. The azoospermia were stratified according to whether DAZ and DAZLA gene locus deletion occurred. Results: DAZ gene locus deletion rate in azoospermia and oligospermia groups was considerably higher than in the control group (P < .05). The DAZLA gene locus deletion rate in the azoospermia group was apparently higher than that in the oligospermia and control groups (P < .05). The semen volume was compared between azoospermia and oligospermia patients and controls (P > .05). Sperm concentration, sperm survival rate, the proportion of normal morphological sperm, and the proportion of progressive motility sperm in the oligospermia group were lower than those in the control group (P < .05). The levels of serum T (Testosterone, T) and T/LH in the azoospermiaspermia group were lower than those in the control group (P < .05). Serum LH (Luteinizing Hormone) and FSH (Follicular Stimulating Hormone) in azoospermia group and oligospermia group were higher than those in the control group (P < .05). The Serum LH determination value of the azoospermia group is higher than the oligospermia group (P < .05). Serum T/LH in the azoospermia group was lower than in the oligospermia group (P < .05). The serum T and T/LH values in azoospermia male patients with DAZ and DAZLA gene deletion were lower than those without deletion (P < .05). Sperm concentration and survival rate of oligospermatism male patients with DAZ gene deletion were lower than those without deletion (P < .05). Sperm and serum T and T/LH values of oligospermatism male patients with DAZ gene deletion were lower than those without deletion (P < .05). Conclusion: The incidence of DAZ and DAZLA gene locus deletion in male patients with idiopathic azoospermia and oligozoospermia was higher than in normal males. The gene locus deletion was related to decreased androgen level, sperm count and motility.

Polyacrylonitrile as an Efficient Transfer Medium for Wafer-Scale Transfer of Graphene.

The disparity between growth substrates and application-specific substrates can be mediated by reliable graphene transfer, the lack of which currently strongly hinders the graphene applications. Conventionally, the removal of soft polymers, that support the graphene during the transfer, would contaminate graphene surface, produce cracks, and leave unprotected graphene surface sensitive to airborne contaminations. In this work, it is found that polyacrylonitrile (PAN) can function as polymer medium for transferring wafer-size graphene, and encapsulating layer to deliver high-performance graphene devices. Therefore, PAN, that is compatible with device fabrication, does not need to be removed for subsequent applications. The crack-free transfer of 4 in. graphene onto SiO2/Si wafers, and the wafer-scale fabrication of graphene-based field-effect transistor arrays with no observed clear doping, uniformly high carrier mobility (≈11 000 cm2 V-1 s-1), and long-term stability at room temperature, are achieved. This work presents new concept for designing the transfer process of 2D materials, in which multifunctional polymer can be retained, and offers a reliable method for fabricating wafer-scale devices of 2D materials with outstanding performance.

Magnesium oxide nanoparticles reduce clubroot by regulating plant defense response and rhizosphere microbial community of tumorous stem mustard (Brassica juncea var. tumida).

Clubroot, caused by Plasmodiophora brassicae, is a major disease that significantly impairs the yield of cruciferous crops and causes significant economic losses across the globe. The prevention of clubroot, especially in tumorous stem mustard (without resistant varieties), are is limited and primarily relies on fungicides. Engineered nanoparticles have opened up new avenues for the management of plant diseases, but there is no report on their application in the prevention of clubroot. The results showed that the control efficacy of 500 mg/L MgO NPs against clubroot was 54.92%. However, when the concentration was increased to 1,500 and 2,500 mg/L, there was no significant change in the control effect. Compared with CK, the average fresh and dry weight of the aerial part of plants treated with MgO NPs increased by 392.83 and 240.81%, respectively. Compared with the F1000 treatment, increases were observed in the content of soil available phosphorus (+16.72%), potassium (+9.82%), exchangeable magnesium (+24.20%), and water-soluble magnesium (+20.64%) in the 1,500 mg/L MgO NPs treatment. The enzyme-linked immune sorbent assay (ELISA) results showed that the application of MgO NPs significantly increased soil peroxidase (POD, +52.69%), alkaline protease (AP, +41.21%), alkaline phosphatase (ALP, +79.26%), urease (+52.69%), and sucrase (+56.88%) activities; And also increased plant L-phenylalanine ammonla-lyase (PAL, +70.49%), polyphenol oxidase (PPO, +36.77%), POD (+38.30%), guaiacol peroxidase (POX, +55.46%) activities and salicylic acid (SA, +59.86%) content. However, soil and plant catalase (CAT, -27.22 and - 19.89%, respectively), and plant super oxidase dismutase (SOD, -36.33%) activities were significantly decreased after the application of MgO NPs. The metagenomic sequencing analysis showed that the MgO NPs treatments significantly improved the α-diversity of the rhizosphere soil microbial community. The relative abundance of beneficial bacteria genera in the rhizosphere soil, including Pseudomonas, Sphingopyxis, Acidovorax, Variovorax, and Bosea, was significantly increased. Soil metabolic functions, such as oxidative phosphorylation (ko00190), carbon fixation pathways in prokaryotes (ko00720), indole alkaloid biosynthesis (ko00901), and biosynthesis of various antibiotics (ko00998) were significantly enriched. These results suggested that MgO NPs might control clubroot by promoting the transformation and utilization of soil nutrients, stimulating plant defense responses, and enriching soil beneficial bacteria.

Effect of n-C-S-H on Hydration and Reinforcement of Mineral Powder-Cement System at Low Temperatures.

This paper investigated the effect of nano-calcium silicate hydrate (n-C-S-H) on the early compressive strength of mineral powder-cement systems under low-temperature curing conditions (5 °C). The hydration mechanism of n-C-S-H in the mineral powder-cement system at different dosages was analyzed by combining it with XRD, DSC-TG, MIP, and other techniques. The results show that n-C-S-H significantly enhances the early compressive strength of the mineral powder-cement system under low-temperature curing conditions, with optimal results observed at a dosage of 1.0% (mass fraction). The XRD, DSC-TG, and MIP tests reveal that n-C-S-H promotes the hydration of the mineral powder cement, accelerates the generation rate of hydration products, reduces the porosity of the hardened mineral powder-cement slurry, and improves the system's density.

Reduction of Backward Scatterings at the Low-Coherence Kunwu Laser Facility.

We report the first experimental observation on the reduction of backward scatterings by an instantaneous broadband laser with 0.6% bandwidth in conditions of interest for inertial confinement fusion at the low-coherence Kunwu laser facility. The backscatter of stimulated Brillouin scattering (SBS) was robustly reduced by half at intensities of 1-5×10^{14} W/cm^{2} with the 0.53-µm broadband laser in comparison with the monochromatic laser. As SBS dominates energy loss of laser-plasma interactions, the reduction of that demonstrates the enhancement of laser-target coupling by the use of broadband laser. The mitigation of filamentation leads to the reduction of stimulated Raman backscattering at low intensities. In addition, the three-halves harmonic emission was reduced with the broadband laser as well.

Experimental confirmation of driving pressure boosting and smoothing for hybrid-drive inertial fusion at the 100-kJ laser facility.

In laser-driven inertial confinement fusion, driving pressure boosting and smoothing are major challenges. A proposed hybrid-drive (HD) scheme can offer such ideal HD pressure performing stable implosion and nonstagnation ignition. Here we report that in the hemispherical and planar ablator targets installed in the semicylindrical hohlraum scaled down from the spherical hohlraum of the designed ignition target, under indirect-drive (ID) laser energies of ~43-50 kJ, the peak radiation temperature of 200 ± 6 eV is achieved. And using only direct-drive (DD) laser energies of 3.6-4.0 kJ at an intensity of 1.8 × 1015 W/cm2, in the hemispherical and planar targets the boosted HD pressures reach 3.8-4.0 and 3.5-3.6 times the radiation ablation pressure respectively. In all the above experiments, significant HD pressure smoothing and the important phenomenon of how a symmetric strong HD shock suppresses the asymmetric ID shock pre-compressed fuel are demonstrated. The backscattering and hot-electron energy fractions both of which are about one-third of that in the DD scheme are also measured.

A water stable and highly fluorescent Zn(II) based metal-organic framework for fast detection of Hg2+, CrVI, and antibiotics.

The development of luminescent metal-organic frameworks for effective sensing and monitoring of environmental pollutants is of great significance for human health and environmental protection. In this work, a novel water-stable ZnII-based luminescent coordination polymer, namely {[Zn(BBDF)(ATP)]·2DMF·3H2O}n ((BBDF = 2,7-bis(1H-benzimidazol-1-yl)-9,9-dimethyl-9H-fluorene) and H2ATP = 2-aminoterephthalic acid), was designed and obtained using the mixed-ligand method. Structural analysis indicated that 1 presents a two-fold interpenetrated two-dimensional layer structure with one dimensional (1D) channels along the a axis. Intriguingly, the uncoordinated -NH2 group was danged onto the pore walls of 1. Remarkably, compound 1 shows good aqueous stability at different pH values of 3-13 and exhibits a fluorescence turn-off sensing behavior for Hg2+, Cr2O72-, CrO42-, and antibiotics (NFZ, NFT) in aqueous solution with high selectivity and sensitivity. The limits of detection (LOD) are 0.12 µM (Hg2+), 0.17 µM (Cr2O72-), 0.21 µM (CrO42-), 0.098 µM (NFZ), and 0.14 µM (NFT). The luminescence quenching mechanism analysis by experiment and theoretical calculation revealed that the competitive absorption and the photoinduced electron transfer process are largely responsible for the sensing of the two antibiotics, while the weak interaction contributes to the selective luminescence quenching for Hg2+.

Mixed-valence {FeII2FeIII4} hexanuclear complexes with thermally induced Fe(III) spin-crossover behavior.

Cyano-bridged mixed-valence {Fe6} hexanuclear complexes {[Tp4-MeFeII(CN)3]2[FeIII(Tpa)]2[FeIII(OR)(Tpa)]2}·6ClO4·S (Tp4-Me = tri(4-methyl-pyrazol-1-yl)borate, Tpa = tris(2-pyridylmethyl)amine; R = -CH3, S = 8MeOH 1, R = -C2H5, S = 6EtOH 2) have been obtained by building the units of [Tp4-MeFe(CN)3]- and [Fe(Tpa)]2+ in methanol and ethanol, respectively. Complex 1 exhibited single-crystal-to-single-crystal (SCSC) transformation to give {[Tp4-MeFeII(CN)3]2[FeIII(Tpa)]2[FeIII(OH)(Tpa)]2}·6ClO4·8H2O (3) in water. Detailed analysis of the coordination environment of the FeIII centers on the square lattice and magnetic susceptibility measurements of all the complexes confirmed their Fe3+ spin-crossover (SCO) properties (T1/2 = 178 K, 1; 185 K, 2; 208 K, 3).

Dual-recognition membrane Adsorbers combining hydrophobic charge-induction chromatography with surface imprinting via multicomponent reaction.

The combination of mixed-mode chromatography (MMC) and molecular imprinting technology (MIT) has been proven to be successful for protein separation, but suffered from cumbersome material preparation and limited performance. In this work, a new modification method marrying atom transfer radical polymerization (ATRP) and multicomponent reaction was proposed to simplify the preparation process. Using regenerated cellulose (RC) membrane as the substrate, immunoglobulin G (IgG) as the template protein and tryptamine as the ligand, a dual-recognition membrane adsorbers (MIM) was prepared by mild Ugi four-component reaction (Ugi-4CR) and surface initiated ATRP. Control the ATRP time is the key for surface imprinting. The static IgG uptake and selectivity of UGI membrane were 45 mg/mL and 1.8, respectively, while those of MIM-0.5 were 42.5 mg/mL and 14, indicating that the introduction of molecular imprinting technology significantly improved the selectivity of the membrane to IgG. The MIM-0.5 membrane retains the pH-dependent and salt-tolerant of HCIC. The dynamic flow-through results showed that the MIM-0.5 membrane could effectively separate IgG from IgG/BSA mixed solution with the purity of 88% and retained its bioactivity. This work demonstrated the feasibility of bonding HCIC and MIT to the membrane surface by Ugi-4CR and ATRP.

Direct generation of relativistic isolated attosecond pulses in transmission from laser-driven plasmas.

Isolated attosecond pulses are useful to perform pump-probe experiments at a high temporal resolution, and provide a new tool for ultrafast metrology. However, it is still a challenging task to generate such pulses of high intensity, even for a few-cycle laser. Through particle-in-cell simulations, we show that it is possible to directly generate a giant isolated attosecond pulse in the transmission direction from relativistic laser-driven plasmas. Compared to attosecond pulse generation in the reflection direction, no further spectral filtering is needed. The underlying radiation mechanism is coherent synchrotron emission, and the transmitted isolated attosecond pulse can reach relativistic intensity. This provides a promising alternative to generate intense isolated attosecond pulses for ultrafast studies.

Cellulose-assisted construction of high surface area Z-scheme C-doped g-C3N4/WO3 for improved tetracycline degradation.

The preparation of heteroatom doping heterojunction photocatalysts with nontoxic carbonaceous materials and simple method still remains a challenge. Herein, ternary Z-scheme C-doped graphitic carbon nitride/tungsten oxide (C-doped g-C3N4/WO3) was successfully fabricated via the hydrothermal impregnation with cellulose nanocrystal, high-temperature calcination, and electrostatic self-assembly with WO3 nanocuboids in turns. Benefiting from the porous structure, high specific areas (57.20 m2 g-1), C-substitution, and the formation of Z-scheme heterojunction, the resulting photocatalyst exhibited narrower band-gap, enhanced visible-light absorption and separation of charge carrier, faster interfacial charge transfer, good oxidation/reduction capacities, and thus improved the photocatalytic activity performance. As such, this investigation will provide an effective route for not only incorporating semiconductors and heteroatoms into g-C3N4 but also developing more heterojunction with markedly improved photocatalytic performance.

One-Pot Hydrothermal Synthesis of La-Doped ZnIn2S4 Microspheres with Improved Visible-Light Photocatalytic Performance.

Impurity element doping is extensively taken as one of the most efficient strategies to regulate the electronic structure as well as the rate of photogenerated charge separation of photocatalysts. Herein, a one-pot hydrothermal synthesis process was exploited to obtain La-doped ZnIn2S4 microspheres, aiming at gaining insight into the role that doping ions played in the improvement of pollutant photodegradation. Systematical characterization means, comprising of X-ray photoelectron spectroscopy (XPS), ultraviolet-visible (UV-vis) diffuse reflection spectroscopy and Raman spectra, combination with high-resolution transmission electron microscopy (HRTEM), were employed to in depth reveal the concomitancy of La ions and ZnIn2S4 crystal lattice. The results showed that the La-doped ZnIn2S4 samples exhibited a slightly wider and stronger spectral absorption than pristine ZnIn2S4; and the specific surface area of doped ZnIn2S4 samples was a bit larger. The La-doped ZnIn2S4 electrodes showed improved photocurrent response, and the photocurrent density reached a maximum value at La content of 1.5 wt%. As expected, La-doped ZnIn2S4 samples exhibited a remarkable enhancement of photocatalytic behaviour toward the photodegradation of tetracycline hydrochloride (TCH) and methyl orange (MO). The prominently enhanced photoactivity of doped ZnIn2S4 samples was due to the synergistic effect of the elevated visible-light absorption ability and effective photogenerated charge carriers' separation.

Lignin-Based Micro- and Nanomaterials and their Composites in Biomedical Applications.

Lignin, as the most abundant aromatic renewable biopolymer in nature, has long been regarded as waste and simply discarded from the pulp and paper industry. In recent years, with many breakthroughs in lignin chemistry, pretreatment, and processing techniques, a lot of the inherent bioactivities of lignin, including antioxidant activities, antimicrobial activities, biocompatibilities, optical properties, and metal-ion chelating and redox activities, have been discovered and this has opened a new field not only for lignin-based materials but also for biomaterials. In this Review, the biological activities of lignin and drug/gene delivery and bioimaging applications of various types of lignin-based material are summarized. In addition, the challenges and limitations of lignin-based materials encountered during the development of biomedical applications are also discussed.

Stimulated Brillouin scattering of backward stimulated Raman scattering.

The rescattering of backward stimulated Raman scattering (BSRS) by stimulated Brillouin scattering (SBS) is found in the high electron density region by relativistic Vlasov-Maxwell simulation and particle-in-cell (PIC) simulation, where the BSRS is in the regime of absolute instability and dominates in all the scatterings. Both one dimension (1D) Vlasov simulation and two dimension (2D) PIC simulation have been given to verify that there exists SBS of BSRS in the regime of absolute instability for BSRS. The SBS of BSRS will be even stronger than forward stimulated Raman scattering (FSRS) and SBS in regime of absolute instability for BSRS. Thus, besides Langmuir decay instability and laser energy absorption, the SBS of BSRS is also an important saturation mechanism of BSRS in high electron density region.

Symmetry Breaking and Two-Step Spin-Crossover Behavior in Two Cyano-Bridged Mixed-Valence {FeIII2(µ-CN)4FeII2} Clusters.

Two new cyano-bridged mixed-valence {FeIII2(µ-CN)4FeII2} clusters, {[(Tp)FeIII(CN)3]2[FeII(Py2N2)]2}·(ClO4)2·MeCN·Et2O (1·MeCN·Et2O), its solvent-free form (1), and {[(Tp)FeIII(CN)3]2[FeII(Me2Py2N2)]2}·(ClO4)2·5MeOH (2·5MeOH), were obtained [Tp = hydrotris(pyrazol-1-yl)borate; N,N'-bis(2-pyridylmethyl)-N,N'-bis(4-X-benzyl)-1,2-ethanediamine, Py2N2, X = H; Me2Py2N2, X = Me]. Complexes 1 and 2·5MeOH exhibit gradual thermally induced two-step spin-crossover behavior (SCO) at two FeII metal centers, and the transformation of high-spin (HS) to low-spin (LS) FeII ions with temperature was confirmed by a combination of X-ray crystallography, variable-temperature Fourier transform infrared, variable-temperature magnetic susceptibility, and 57Fe Mössbauer spectroscopy. Moreover, complexes 1·MeCN·Et2O and 1 exhibit a reversible single-crystal-to-single-crystal transformation, and complex 1 undergoes two-step SCO behavior with T1/2 = 178 and 93 K accompanied by symmetry breaking in the structure.

Pharmacological modulation of autophagy as a novel potential target in the successful implementation of in vitro fertilization.

Autophagy is an important intracellular process to maintain homeostasis and studies have shown the key role of autophagy in modulating the functions of reproductive system. Alongside with it, the activation of autophagy has also been found to regulate a number of important processes involved in in vitro fertilization including degeneration of granulosa cells and oocyte defects in obese and aging women; apoptosis of oocytes during vitrification-warming; quality and viability of embryo; developmental competence and pre-implantation development of in vitro produced blastocysts; placental vascularization and fetal growth. The different mechanisms that may contribute in autophagy-mediated increase in developmental competence and pre-implantation development include decrease in endoplasmic reticulum (ER) stress, activation of poly(ADP-ribosyl)ation (PARP) and reduction in free radical production. The present review discusses the role of autophagy activation in increasing the efficiency of in vitro fertilization by modulating different aspects related to fertilization.

Identification of a Quorum Sensing System Regulating Capsule Polysaccharide Production and Biofilm Formation in Streptococcus zooepidemicus.

Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well-known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.

Experimental demonstration of low laser-plasma instabilities in gas-filled spherical hohlraums at laser injection angle designed for ignition target.

Octahedral spherical hohlraums with a single laser ring at an injection angle of 55^{∘} are attractive concepts for laser indirect drive due to the potential for achieving the x-ray drive symmetry required for high convergence implosions. Laser-plasma instabilities, however, are a concern given the long laser propagation path in such hohlraums. Significant stimulated Raman scattering has been observed in cylindrical hohlraums with similar laser propagation paths during the ignition campaign on the National Ignition Facility (NIF). In this Rapid Communication, experiments demonstrating low levels of laser-driven plasma instability (LPI) in spherical hohlraums with a laser injection angle of 55^{∘} are reported and compared to that observed with cylindrical hohlraums with injection angles of 28.5^{∘} and 55^{∘}, similar to that of the NIF. Significant LPI is observed with the laser injection of 28.5^{∘} in the cylindrical hohlraum where the propagation path is similar to the 55^{∘} injection angle for the spherical hohlraum. The experiments are performed on the SGIII laser facility with a total 0.35-µm incident energy of 93 kJ in a 3 nsec pulse. These experiments demonstrate the role of hohlraum geometry in LPI and demonstrate the need for systematic experiments for choosing the optimal configuration for ignition studies with indirect drive inertial confinement fusion.

Spin crossover and reversible single-crystal to single-crystal transformation behaviour in two cyanide-bridged mixed-valence {FeFe} clusters.

The reactions of tricyanometallate precursors, (Ph3PMe)[(Tp4-Me)Fe(CN)3·0.5CH3CN] (1) (Tp4-Me = tri(4-methyl-pyrazol-1-yl)borate) and (NBu4)[(MeTp)Fe(CN)3] (MeTp = methyltris(pyrazolyl)borate) with the presence of the tetradentate tpa ligand (tpa = tris(2-pyridylmethyl)amine) and Fe(ClO4)2·6H2O afford two new cyano-bridged mixed-valence {FeFe} molecular squares: [(Tp4-Me)FeIII(CN)3]2[FeII(tpa)]2·2ClO4·H2O (2·H2O) and [(MeTp)FeIII(CN)3]2[FeII(tpa)]2·2ClO4·CH3OH (3·CH3OH). Solvent-exchange compounds of 3·CH3OH, [(MeTp)FeIII(CN)3]2[FeII(tpa)]2·2ClO4·2H2O (3·2H2O), and their solvent-free form (2 and 3) are also obtained, respectively. The spin crossover (SCO) properties of all compounds are confirmed by detailed structural analyses of the coordination environments of the FeII centres and magnetic susceptibility measurements. All compounds exhibit SCO behaviour near room temperature (T1/2 = 320 K for 2·H2O; 302 K for 2; 292 K for 3·CH3OH; 306 K for 3·2H2O and 290 K for 3) and reversible single-crystal to single-crystal (SC-SC) transformations induced by guest desorption and resorption or solvent exchange. The transition temperature close to room temperature can be tuned by the dehydration and re-hydration processes. The structure-property analysis discloses that the distorted {Fe4(µ-CN)4} core and deviations of bent ∠Fe-N[triple bond, length as m-dash]C angles play a key role in tuning the transition temperature of these similar mixed-valence {FeFe} complexes.

Factors Impacting Electron Transfer in Cyano-Bridged {Fe2Co2} Clusters.

A cyano-bridged {Fe(III)2Co(II)2} complex exhibits reversible thermally and photoinduced intramolecular charge transfer. Its desolvated, MeOH-d4, and other analogues were compared to disclose the impact factors on the electron-transfer behavior of these {Fe(III)2Co(II)2} clusters.