Cofactors are essential components of numerous enzymes, therefore their characterization by structural, biophysical, and biochemical approaches is crucial for understanding the resulting catalytic and regulatory mechanisms. In this chapter, we present a case study of a recently discovered cofactor, the nickel-pincer nucleotide (NPN), by demonstrating how we identified and thoroughly characterized this unprecedented nickel-containing coenzyme that is tethered to lactase racemase from Lactiplantibacillus plantarum. In addition, we describe how the NPN cofactor is biosynthesized by a panel of proteins encoded in the lar operon and describe the properties of these novel enzymes. Comprehensive protocols for conducting functional and mechanistic studies of NPN-containing lactate racemase (LarA) and the carboxylase/hydrolase (LarB), sulfur transferase (LarE), and metal insertase (LarC) used for NPN biosynthesis are provided for potential applications towards characterizing enzymes in the same or homologous families.

Sulfur-insertion reactions are essential for the biosynthesis of several cellular metabolites, including enzyme cofactors. In Lactobacillus plantarum, a sulfur-containing nickel-pincer nucleotide (NPN) cofactor is used as a coenzyme of lactic acid racemase, LarA. During NPN biosynthesis in L. plantarum, sulfur is transferred to a nicotinic acid-derived substrate by LarE, which sacrifices the sulfur atom of its single cysteinyl side chain, forming a dehydroalanine residue. Most LarE homologs contain three conserved cysteine residues that are predicted to cluster at the active site; however, the function of this cysteine cluster is unclear. In this study, we characterized LarE from Thermotoga maritima (LarETm) and show that it uses these three conserved cysteine residues to bind a [4Fe-4S] cluster that is required for sulfur transfer. Notably, we found LarETm retains all side chain sulfur atoms, in contrast to LarELp. We also demonstrate that when provided with L-cysteine and cysteine desulfurase from Escherichia coli (IscSEc), LarETm functions catalytically with IscSEc transferring sulfane sulfur atoms to LarETm. Native mass spectrometry results are consistent with a model wherein the enzyme coordinates sulfide at the nonligated iron atom of the [4Fe-4S] cluster, forming a [4Fe-5S] species, and transferring the noncore sulfide to the activated substrate. This proposed mechanism is like that of TtuA that catalyzes sulfur transfer during 2-thiouridine synthesis. In conclusion, we found that LarE sulfur insertases associated with NPN biosynthesis function either by sacrificial sulfur transfer from the protein or by transfer of a noncore sulfide bound to a [4Fe-4S] cluster.

The coronavirus disease 2019 (COVID-19) pandemic has posed a threat to public health throughout the world since the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was discovered in late 2019. Since the beginning of the pandemic, scientists have done a tremendous amount of work in this area. However, among these studies, the investigation of the effect of newly synthesized compounds against coronavirus is rather weak. Examining the newly synthesized compounds with a computer-assisted molecular docking study provides quite an advantage in terms of the estimation and analysis of the biochemical activity and binding affinity of existing synthesized compounds against a biological target in a labor, time, and cost-saving way. In this study, the SNS pincer type 2,6-bis[[(4-methylphenyl)thio]methyl]pyridine ligand(L) (1) and its novel Pd(II) complexes ([Pd(κ2-L)(OAc)2]·3H2O (2) and [Pd(κ2-L)Cl2]·3H2O (3)) were synthesized and characterized by using FT-IR, UV-Vis, NMR, mass and elemental analysis techniques. The synthesized Pd complexes exhibited a square planar structure. The compounds were found to have non-electrolytic behavior. In the meantime, in silico investigations have defined and justified interaction processes between these molecules and Pd(II) at the atomic level. Furthermore, using molecular docking against target proteins of SARS-CoV-2, the efficiency of the SNS pincer type ligand and its Pd (II) complexes produced was studied and discussed for the first time. The experimental data has been supported and illuminated using computational visual methods and molecular docking, and the findings produced indicate compatibility. The binding energy values of the relevant compounds on the four protein model structures of SARS-CoV-2 (Main Protease, Papain-like protease, RdRp without RNA, and RdRp with RNA) are represented. Compound 2 ([Pd(κ2-L)(OAc)2]·3H2O) is the structure that exhibits the highest biochemical activity. According to all of the docking studies, Papain-like protease is the SARS-CoV-2 protein with which the three compounds exhibit mutual interaction. The compound 2 structure, in particular, is the most effective in terms of structural and interaction with the targets, as well as binding orientations.

UNASSIGNED: Femoroacetabular impingement (FAI) is the result of abnormal contact between the proximal femur and the acetabulum rim. Pincer impingement is the result of a globally deep acetabulum, focal anterior overcoverage or less commonly posterior overcoverage. The presence of radiological signs of femoroacetabular impingement (FAI) is not necessarily associated with symptoms. The study aims to find the prevalence of such signs in asymptomatic adult individuals.
UNASSIGNED: Data of 714 patients (1428 hips) who have undergone PBH-AP (Pelvis Both hip- Anterior-Posterior view) radiograph for conditions unrelated to the disorders of the hip were taken for study. We evaluated the images for the presence of a cross-over sign (COS) and ischial spine sign (ISS) and measured Lateral Central-Edge (LCE) angle (Wiberg) and Tonnis angle (TA).
UNASSIGNED: Mean age was 33.4 ± 9.8 years. Positive cross-over signs and ischial spine signs were seen in 26.6% and 13.9%, respectively; LCE angle > 40° in 25.5% and Tonnis angle ≤ 0° in 13.5% patients. 36.7% of patients showed atleast one radiological sign of acetabulum overcoverage. There was a significant difference in means of the values of LCE angle and Tonnis angle between males and females (2.9 and - 1.2, respectively). There was no significant difference in prevalence of COS and ISS among the two genders.
UNASSIGNED: This study shows that there is higher prevalence of radiographic signs (36.7%) of pincer deformity among asymptomatic adults. Therefore, new clinical studies are required to clarify the actual significance of these findings.

UNASSIGNED: Accurate assessment of osseous morphology is imperative in the evaluation of patients with femoroacetabular impingement syndrome (FAIS) and hip dysplasia. Through use of computed tomography (CT), 3-dimensional (3D) reconstructed hip models may provide a more precise measurement for overcoverage and undercoverage and aid in the interpretation of 2-dimensional radiographs obtained in the clinical setting.
UNASSIGNED: To describe new measures of acetabular coverage based on 3D-reconstructed CT scan bone models.
UNASSIGNED: Cross-sectional study; Level of evidence, 3.
UNASSIGNED: Preoperative CT scans were acquired on the bilateral hips and pelvises of 30 patients before arthroscopic surgical intervention for FAIS. Custom software was used for semiautomated segmentation to generate 3D osseous models of the femur and acetabulum that were aligned to a standard coordinate system. This software calculated percentage of total acetabular coverage, which was defined as the surface area projected onto the superior aspect of the femoral head. The percentage of coverage was also quantified regionally in the anteromedial, anterolateral, posteromedial, and posterolateral quadrants of the femoral head. The acetabular clockface was established by defining 6 o\'clock as the inferior aspect of the acetabular notch. Radial coverage was then calculated along the clockface from the 9-o\'clock to 5-o\'clock positions.
UNASSIGNED: The study included 20 female and 10 male patients with a mean age of 33.6 ± 11.7 years and mean body mass index of 27.8 ± 6.3. The average percentage of total acetabular coverage for the sample was 57% ± 6%. Acetabular coverages by region were as follows: anteromedial, 78% ± 7%; anterolateral, 18% ± 7%, posterolateral, 33% ± 13%, and posteromedial, 99% ± 1%. The acetabular coverage ranged from 23% to 69% along the radial clockface from 9 to 5 o\'clock.
UNASSIGNED: This study demonstrated new 3D measurements to characterize acetabular coverage in patients with FAIS and elucidated the distribution of acetabular coverage according to these measurements.

To examine whether acetabular dysplasia (AD), cam and/or pincer morphology are associated with radiographic hip osteoarthritis (rHOA) and hip pain in UK Biobank (UKB) and, if so, what distribution of osteophytes is observed.
Participants from UKB with a left hip dual-energy X-ray absorptiometry (DXA) scan had alpha angle (AA), lateral centre-edge angle (LCEA) and joint space narrowing (JSN) derived automatically. Cam and pincer morphology, and AD were defined using AA and LCEA. Osteophytes were measured manually and rHOA grades were calculated from JSN and osteophyte measures. Logistic regression was used to examine the relationships between these hip morphologies and rHOA, osteophytes, JSN, and hip pain.
6,807 individuals were selected (mean age: 62.7; 3382/3425 males/females). Cam morphology was more prevalent in males than females (15.4% and 1.8% respectively). In males, cam morphology was associated with rHOA [OR 3.20 (95% CI 2.41-4.25)], JSN [1.53 (1.24-1.88)], and acetabular [1.87 (1.48-2.36)], superior [1.94 (1.45-2.57)] and inferior [4.75 (3.44-6.57)] femoral osteophytes, and hip pain [1.48 (1.05-2.09)]. Broadly similar associations were seen in females, but with weaker statistical evidence. Neither pincer morphology nor AD showed any associations with rHOA or hip pain.
Cam morphology was predominantly seen in males in whom it was associated with rHOA and hip pain. In males and females, cam morphology was associated with inferior femoral head osteophytes more strongly than those at the superior femoral head and acetabulum. Further studies are justified to characterise the biomechanical disturbances associated with cam morphology, underlying the observed osteophyte distribution.

Two penta-coordinated [Co(MorphBPT)Cl2]; 1 and [Co(PipBPT)Cl2]; 2 complexes with the bis-pyrazolyl-s-triazine pincer ligands MorphBPT and PipBPT were synthesized and characterized. Both MorphBPT and PipBPT act as NNN-tridentate pincer chelates coordinating the Co(II) center with one short Co-N(s-triazine) and two longer Co-N(pyrazole) bonds. The coordination number of Co(II) is five in both complexes, and the geometry around Co(II) ion is a distorted square pyramidal in 1, while 2 shows more distortion. In both complexes, the packing is dominated by Cl…H, C-H…π, and Cl…C (anion-π stacking) interactions in addition to O…H interactions, which are found only in 1. The UV-Vis spectral band at 564 nm was assigned to metal-ligand charge transfer transitions based on TD-DFT calculations. Complexes 1 and 2 showed higher antimicrobial activity compared to the respective free ligand MorphBPT and PipBPT, which were not active. MIC values indicated that 2 had better activity against S. aureus, B. subtilis, and P. vulgaris than 1. DPPH free radical scavenging assay revealed that all the studied compounds showed weak to moderate antioxidant activity where the nature of the substituent at the s-triazine core has a significant impact on the antioxidant activity.

The reaction chemistry of the bis-tetrazinyl pyridine ligand (btzp) towards nitrogen oxyanions coordinated to zinc is studied in order to explore the reduction of the NOx - substrates with a redox-active ligand in the absence of redox activity at the metal. Following syntheses and characterization of (btzp)ZnX2 for X=Cl, NO3 and NO2 , featuring O-Zn linkage of both nitrogen oxyanions, it is shown that a silylating agent selectively delivers silyl substituents to tetrazine nitrogens, without reductive deoxygenation of NOx -1 . A new synthesis of the highly hydrogenated H4 btzp, containing two dihydrotetrazine reductants is described as is the synthesis and characterization of (H4 btzp)ZnX2 for X=Cl and NO3 , both of which show considerable hydrogen bonding potential of the dihydrotetrazine ring NH groups. The (H4 btzp)ZnCl2 complex does not bind zinc in the pincer pocket, but instead H4 btzp becomes a bridge between neighboring atoms through tetrazine nitrogen atoms, forming a polymeric chain. The reaction of AgNO2 with (H4 btzp)ZnCl2 is shown to proceed with fast nitrite deoxygenation, yielding water and free NO. Half of the H4 btzp reducing equivalents form Ag0 and thus the chloride ligand remains coordinated to the zinc metal center to yield (btzp)ZnCl2 . To compare with AgNO2 , experiments of (H4 btzp)ZnCl2 with NaNO2 result in salt metathesis between chloride and nitrite, highlighting the importance of a redox-active cation in the reduction of nitrite to NO.

UNASSIGNED: Radiographic findings related to the cam and pincer variants of femoroacetabular impingement (FAI) include measurements of the alpha angle and lateral center-edge angle (LCEA). The function of these radiographic findings has been put into question because of high heterogeneity in reported studies.
UNASSIGNED: The aim of this study was 3-fold: (1) to determine the prevalence of cam and pincer variants according to sex and age on anteroposterior (AP) pelvic radiographs from an asymptomatic nonathletic population, (2) to identify the most common radiographic signs of cam- and pincer-type variants, and (3) to determine if there are variations in the prevalence of these radiographic signs according to sex and age.
UNASSIGNED: Cross-sectional study; Level of evidence, 3.
UNASSIGNED: There were 3 independent observers who retrospectively analyzed the 939 AP pelvic radiographs (1878 hips) of patients aged 18 to 50 years who did not have hip symptoms and who were not professional athletes. The prevalence of the cam and pincer variants according to the alpha angle and LCEA, respectively, and the presence of other radiographic signs commonly associated with these variables were determined in the overall population and by subgroup according to sex and age group (18-30, 31-40, and 41-50 years). Descriptive and inferential statistics were used to analyze the study sample.
UNASSIGNED: The mean age of the included population was 31.0 ± 9.2 years, and 68.2% were male. The prevalence of the cam-type variant was 29.7% (558/1878), and that of the pincer-type variant was 24.3% (456/1878). The radiographic signs that were most associated with the cam and pincer variants were a pistol-grip deformity and the crossover sign, respectively. Significant differences (P < .001) in the prevalence of these variants were identified between men and women in both variants. No differences were observed in the alpha angle or LCEA according to sex or age.
UNASSIGNED: Radiographic findings suggestive of FAI had significant variations with respect to sex and age in this study sample. This study provides information to determine the prevalence of these anatomic variants in the general population.

UNASSIGNED: Although the association between femoroacetabular impingement (FAI) syndrome and hip osteoarthritis (OA) is well established, not all hips exhibiting cam or pincer morphologies (i.e. imaging findings of FAI syndrome) are symptomatic or arthritic. It is difficult to detect which subgroup will wear out, or how does the arthritic process start radiographically. Therefore, we measured in a retrospective study based on computed tomography (CT) analysis, the joint space width (JSW) according to a standard protocol and we investigated its variation according to the presence of a cam and/or pincer morphology. We hypothesized that the radiological presence of a cam and/or pincer hip morphologies, even in asymptomatic subjects, would affect JSW.
UNASSIGNED: Two hundred pelvic CT scans performed for non-orthopedic etiologies in asymptomatic patients were analyzed using a 3D software. After excluding patients with hip OA or previous hip surgery, 194 pelvic CT scans (388 hips) were retained. We measured for each hip the presence of FAI syndrome imaging findings (cam and pincer morphologies) using the classical parameters of coxometry. In addition, we performed a measurement of articular joint space width according to a standard protocol. We then calculated the mean thickness of 3 defined regions along the femoroacetabular joint: anterior-superior, posterior-inferior, and posterior-superior. Lastly, we compared the JSW across 4 groups: hips with (1) no cam or pincer, (2) pincer, (3) cam, and (4) cam and pincer morphologies using a multivariate analysis. Additionally, a topographic heatmap of JSW was plotted allowing quantitative representation of JSW along the joint.
UNASSIGNED: Increased JSW with peak difference of 0.9 mm (25.7%) was found in hips with cam and pincer morphologies when compared to normal ones (p = 0.002) and to hips with pincer or cam morphologies only.
UNASSIGNED: Positive variations in JSW were associated to the presence of cam and pincer morphologies. This significant increase in JSW could be one of the earliest measurable changes preceding later classical alterations.