Alpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies that pathologically define a group of disorders known as synucleinopathies, including Parkinson’s Disease (PD) and Dementia with Lewy Bodies. αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal counts in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and reduction in NECAB1-positive neuron counts induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced reduction in tyrosine hydroxylase (TH)-positive dopaminergic cells in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and enhanced PFF-induced reduction in TH-positive dopaminergic cells. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo. Alpha-synuclein (αsyn) is a critical protein whose aggregation and transmission from cell to cell has been implicated in the neurodegenerative process in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is a highly expressed brain protein whose endogenous function is not well understood but likely includes regulation of synaptic transmission –3.ThoroGiven 14-3-3s’ roles in protein folding and trafficking, we recently examined the impact of 14-3-3s on αsyn cell-to-cell transmission and toxicity in two separate cellular models: the paracrine αsyn model and the in vitro αsyn fibril model [Mice were used in accordance with the guidelines of the National Institute of Health (NIH) and University of Alabama at Birmingham (UAB) Institutional Animal Care and Use Committee (IACUC). All animal work performed in this study was approved by UAB’s IACUC. Transgenic mice expressing human 14-3-3θ under the neuronal promoter Thy1.2 were previously developed by our group . TransgeRecombinant full-length mouse αsyn protein was prepared as previously described and generously supplied by Dr. Laura Volpicelli-Daley and Dr. Andrew West , 22. BefAAV2/CBA-IRES2-eGFP-WPRE (AAV-GFP) and AAV2/CBA-14-3-3θ-V5-his-IRES-eGFP-WPRE (AAV-14-3-3θ) were constructed as previously described , 23. EquEqual numbers of male and female mice at 8–12 weeks of age were deeply anesthetized with 5% isoflurane and maintained at 0.25–4% during surgery for PFF injection. WT mice injected with AAV-GFP or AAV-14-3-3θ/GFP at 8 weeks of age were then injected at 12 weeks of age with PFFs. 14-3-3θ, difopein, or WT littermates were injected at 8–12 weeks of age with PFFs. Mice were unilaterally injected with 5 µg of αsyn fibrils or monomer (2 µl of 2.5 mg/ml) at a flow rate of 0.250 µl/minute into the dorsolateral striatum , according to a previously established protocol . Post-su2 area.Mice were perfused with PBS followed by 4% paraformaldehyde using a forced pump system. After dissection, brains were sliced by microtome in 40 µm thick coronal sections. Every sixth section from the anterior brain  was stained for pS129 αsyn or NECAB1 immunohistochemistry, while every fourth section from the posterior sections (including the SN) was stained for pS129 αsyn or TH immunohistochemistry. For pS129 αsyn and TH immunohistochemistry, sections were quenched with 0.6% hydrogen peroxide in methanol followed by antigen retrieval  for 1 h at 37° C. Sections were then blocked in 5% normal goat serum (NGS) with 0.3% Triton X-100 and incubated for 48 h in pS129-αsyn antibody (Abcam #51253) or 24 h in TH antibody (Pelfreez #40101) in 1.5% NGS at 4 °C. After washing, sections were incubated with goat anti-rabbit IgG biotinylated secondary antibody (Vector Laboratories #BA-1000) for 4 h at 4 °C. After washing with TBS, sections were incubated in ABC solution (Vector) for 30 min and developed using ImmPACT DAB Chromagen Solution (Vector). Following washing with TBS, sections were mounted on slides and progressively dehydrated with ethanol and Histo-clear. Slides were cover-slipped with Permount (Avantar) and imaged using an Olympus BX51 epifluorescence microscope. For pS129-αsyn positive inclusion counts, 3 sections in either the sensorimotor cortical regions (Bregma + 1.54 mm to + 0.62 mm) or the SN (Bregma − 2.70 mm to − 3.88 mm) per well were selected and quantitated using ImageJ with the rater blind to experimental conditions. One section was selected in the central and basolateral amygdala region (Bregma − 0.82 mm to − 1.34 mm) and quantitated using ImageJ with the rater blind to experimental conditions. αSyn inclusion counts were normalized per mm2 area.For fluorescence staining, sections were washed in TBS followed by antigen retrieval  for 1 h at 37 °C. Sections were then blocked in 5% NGS with 0.1% Triton X-100 and incubated with anti-NECAB1 rabbit antibody (Sigma Millipore #HPA023629), anti-NeuN mouse antibody (Thermofisher Scientific #14H6L24 or Sigma Millipore #MAB377), anti-GFAP mouse antibody , or pS129-αsyn rabbit antibody (Abcam #51253) overnight at 4 °C. After washing with TBS, sections were incubated with Cy3-conjugated goat anti-mouse secondary antibody and Alexa488-conjugated goat anti-rabbit antibody for 2 h at 4 °C. Sections were mounted on slides, cover-slipped using ProLong Diamond Antifade mounting solution (Thermofisher Scientific), and imaged using an Olympus BX51 epifluorescence microscope or a Nikon Eclipse Ti2 confocal microscope. For NECAB1-positive neuronal counts, 3 sections in the sensorimotor cortical regions (Bregma + 1.54 mm to + 0.62 mm) per well were selected and quantitated using ImageJ with the rater blind to experimental conditions. NECAB1 counts were normalized to mmStereological estimates of TH-positive neuronal numbers were performed using the optical fractionator method of the StereoInvestigator 8.0 software from MBF Biosciences , as previously described , 25. ForBehavior tests to assess motor and social functions were conducted 3 and 6 months after PFF injection. Mice were handled for 3 to 5 days before testing began and habituated to testing room for 30 min at the start of each testing day. Behavior tests began at least 1 h after light/dark cycle switch and completed at least 1 h before switching back to their dark cycle. In order to minimize stress to the animals, behavior tests were ordered from least to most stressful as follows: open field, wire hang, social dominance (tube test), pole test, and finally rotarod. Behavior was done primarily at 6 months post injection (mpi), but the difopein cortical cohort did undergo behavioral tests at 3 mpi. All behavior apparatuses were cleaned with 2% chlorohexidine between trials in order to minimize scent contamination between mice. Further, male mice were tested before female mice to minimize their exposure to female scent.For open field, mice were placed with a 48 in. × 48 in. open arena with clear plexi-glass walls. Mice were videoed for 4 min using EthoVision software and analyzed for overall velocity, distance moved across all planes , and time spent in the periphery.A four limb wire hang test was performed, as previously described with some modifications . An appa. [For the tube test, mice were placed in a clear 12″ long tube at opposing ends as previously described in protocols established by Arrant et al. , 28. MicAt 3 mpi (aged 5–6 months), mice were placed facing upwards at the top of a ¼″ diameter round 3-foot long pole. Each mouse was timed for the duration to turn downwards down the pole as well as descend to the bottom of the pole. At 6 mpi (mouse age 8–9 months), wire footholds were added to the pole to assist mice in descending safely due to age and weight gain.To further assess motor activity at 6 mpi, mice were placed on an accelerating rotarod apparatus with speed increasing from 5 to 35 rpm over 60 s. Mice were given 3 days of training on the apparatus and 2 days of testing. All mice underwent 3 trials per day with an inter-trial rest time of 2 min on training days and 5 min on assessment days. No mice failed to fall during the allotted 60 s.p ≤ 0.05. All the details of experiments can be found in the results section or figure legends. All data values are presented as mean ± SEM. ANOVA related statistics  are noted in the results section, while the post-hoc test results are found in the figure legends. For t-tests, the t statistic and p values are noted in the results section.GraphPad Prism 8  was used for statistical analysis of experiments. Data were analyzed by either Student’s t-test, one-way ANOVA, or two-way ANOVA, followed by post-hoc pairwise comparisons using Tukey’s multiple comparison tests. Statistical significance was set at , we examined the effect of 14-3-3θ overexpression or 14-3-3 inhibition on behavioral deficits, αsyn inclusion formation, and neuronal counts in the in vivo fibril model  = 1.079, p = 0.3044; interaction F  = 0.008179, p = 0.9283) or distance traveled  = 0.2940, p = 0.5903; PFF treatment F  = 1.088, p = 0.3024; interaction F  = 0.007373, p = 0.9320) between WT and 14-3-3θ transgenic mice, whether injected with monomeric or fibrillar αsyn  = 0.5662, p = 0.4557; interaction F  = 0.6048, p = 0.4408; Additional file p = 0.9963; PFF treatment F  = 0.01661, p = 0.8980; interaction F  = 0.08812, p = 0.7680; Additional file p = 0.7781; PFF treatment F  = 2.507, p = 0.1203; interaction F  = 0.02684; p = 0.8706; Additional file Previous studies have shown motor and/or cognitive behavioral effects in response to αsyn PFF injection into the dorsolateral striatum , 28, 29.ent F 1, 5 = 1.079ent F 1, 5 = 1.079ent F 1, 5 = 1.079ent F 1, 5 = 1.079ent F 1, 5 = 1.079p = 0.0060; Fig. We did observe a strong effect of PFF injections in WT animals in the social dominance test, which is thought to be a measure of prefrontal cortical and amygdala function , 30–33, p = 0.8263; PFF treatment F  = 10.01, p = 0.0026, interaction  = 0.2352, p = 0.6297) and on the pole test  = 0.02336, p = 0.8791; PFF treatment F  = 4.251, p = 0.0442; interaction F  = 0.7770, p = 0.3821), no significant differences were observed between GFP mice injected with monomer vs. GFP mice injected with PFFs or between GFP mice injected with PFFs and 14-3-3θ mice injected with PFFs on either pole test or open field testing at 6 mpi  = 2.342, p = 0.1320; interaction F  = 0.2655, p = 0.6086; Additional file Since our transgenic 14-3-3θ line does not demonstrate 14-3-3θ overexpression in the nigra  = 10.57, p = 0.0020; interaction F  = 0.2741, p = 0.6027) and distance traveled  = 0.1289, p = 0.7209; PFF effect F  = 11.94, p = 0.0011; interaction F  = 0.6675, p = 0.4174) in mice on the open field test, but no dramatic differences were noted between individual experimental groups at 6 mpi  = 0.6636; p = 0.4187; interaction F  = 0.9522, p = 0.3333; Additional file We next examined whether inhibition of 14-3-3s with the pan-14-3-3 peptide inhibitor difopein affects behavioral deficits in the in vivo PFF model. Transgenic mice expressing difopein in the cortex showed a deficit in social dominance after PFF injection that was not observed in WT mice at 3 mpi  = 3.139, p = 0.4108; PFF treatment F  = 0.2927, p = 0.5905; interaction F  = 5.109; p = 0.0274) or velocity  = 0.2982, p = 0.5870; PFF treatment F  = 0.03472, p = 0.8528; interaction F  = 13.13, p = 0.0006) on the open field test at 6 mpi, although a slight interaction effect was noted  = 2.386, p = 0.1281; interaction F  = 0.09658, p = 0.7571; Additional file p).Similar to that observed with the AAV-14-3-3θ injections into the SN, expression of difopein in the SN did not impact motor function significantly. PFF or genotype did not impact distance traveled  = 0.6861, (13) = 2.754, p = 0.0164; Fig. (17) = 3.232, p = 0.0049; Fig. (15) = 2.757, p = 0.0147) but then a 60% increase in the number of inclusions in the amygdala at 6 mpi  = 2.193, p = 0.0471) in 14-3-3θ transgenic mice injected with PFFs compared to WT mice injected with PFFs  = 0.1161, p = 0.9094) or 6 mpi  = 0.5746, p = 0.5782) in the SN, in which 14-3-3θ overexpression is not seen in these transgenic mice  in the sensorimotor cortex of WT mice injected with PFFs  = 2.229, p = 0.0350 Fig. (25) = 2.481, p = 0.0202; Fig. Since our transgenic 14-3-3θ line does not demonstrate 14-3-3θ overexpression in the SN, we also examined nigral inclusion formation in the mice stereotactically injected with AAV-GFP or AAV-14-3-3θ/GFP in the SN  = 2.441, p = 0.0210; Fig. (25) = 2.233, p = 0.0347; Fig. (21) = 2.070, p = 0.05) but showed a non-significant decrease (53%) at 6 mpi  = 2.021, p = 0.0616) in difopein mice compared to WT mice  = 1.123, p = 0.2804) and decreased significantly at 6 mpi  = 2.355, p = 0.0283) after PFF injection compared to WT mice  = 10.01, p = 0.0029; interaction F  = 2.514, p = 0.1202; Fig. As noted above, αsyn inclusion numbers are much higher at 3 months after PFF injection than at 6 months after injection in WT animals. This reduction in αsyn inclusion numbers over time is presumably secondary to the death of neurons that develop αsyn inclusions . 14-3-3θp = 0.0798; PFF treatment F  = 58.27, p < 0.0001; interaction F  = 4.309, p = 0.0435; Fig. p = 0.0816; PFF treatment F  = 0.3335, p = 0.5686; interaction F  = 1.105, p = 0.3028; Fig. We next examined TH-positive neuronal counts in response to PFFs in difopein-expressing mice. Stereological analysis of TH-positive counts in the nigra revealed a greater reduction in ipsilateral dopaminergic neuronal counts in PFF-injected mice expressing difopein in the SN compared to WT mice after PFF injection at 6 mpi  = 3.208, p = 0.2428; PFF treatment F  = 35.68, p < 0.0001; interaction F  = 4.681, p = 0.0395; Additional file To assess potential neuronal loss in the cortex of WT and transgenic difopein mice, we measured counts of layer IV pyramidal neurons. Since inclusion formation primarily occurs in layer IV and V pyramidal neurons , we usedIn this study we examined the role of 14-3-3s on αsyn aggregation and neuronal toxicity in the in vivo pre-formed fibril (PFF) mouse model. We tested the effects of 14-3-3θ overexpression and 14-3-3 inhibition by the pan-peptide inhibitor difopein in either the cortex or the SN at 2 time points after PFF injection. 14-3-3θ overexpression in the cortex and amygdala, as demonstrated in our 14-3-3θ transgenic line, delayed αsyn inclusion formation and rescued the social dominance deficit. 14-3-3θ overexpression in the SN by AAV also delayed αsyn inclusion formation and partially rescued reductions in TH-positive neuronal counts. In contrast, difopein expression accelerated αsyn inclusion formation, the social dominance behavioral deficit, and reductions in TH-positive and NECAB1-positive neuronal counts. These results suggest that 14-3-3s act as a chaperone to reduce αsyn aggregation and its resulting toxicity on behavioral function and neuronal populations.We and other groups have demonstrated that inclusion formation in the mouse PFF model peaks around 3 months after PFF injection with a subsequent reduction in inclusions at later time points , 34. ThipS129-αsyn positive inclusions were predominantly localized in neurons, suggesting a neuronal selective vulnerability in this model. Previous groups have established that genetic αsyn mouse models with or without PFF treatment can demonstrate the formation of inclusions in glial populations, such as astrocytes, particularly at late stages –42. Few In order to assess the effects of 14-3-3s on αsyn toxicity in the PFF model, we used both transgenic and AAV-induced expression mouse models. Reproducible and consistent findings between AAV and transgene methods of 14-3-3θ overexpression suggest that our findings are not an artifact of transgene expression. The use of both methods also allowed for the assessment of selective areas of expression in modulating the effects of αsyn after initial aggregation initiated by PFF injection in the striatum, as αsyn inclusion formation is seen throughout multiple brain regions in both PD patients and in the PFF model. Our findings demonstrate that 14-3-3s’ effects on αsyn are not restricted to particular brain regions, but that 14-3-3s can impact αsyn pathology in multiple areas in which αsyn pathology is observed in the PFF model. Of note, we tested the effects of the overexpression of a single isoform 14-3-3θ in comparison to pan inhibition of 14-3-3 isoforms by difopein. Our lab has previously established the integral role of 14-3-3θ in modulating αsyn spread and toxicity . We used14-3-3 proteins interact with multiple aggregation-prone proteins in neurodegenerative diseases, including tau, huntingtin, and αsyn –49. Our Based on our findings here and in other studies, we propose that disruption of 14-3-3 function may serve to promote the neurodegenerative process in PD and DLB. Alterations in 14-3-3s have been noted in PD models and in human disease , 56. We Motor phenotypes in the PFF model have been variably reported, with some groups finding strong motor deficits and others observing none , 28, 29.In conclusion, we found that 14-3-3θ overexpression reduced behavioral deficits, delayed αsyn aggregation, and partially prevented decreases in TH-positive neuronal counts, while 14-3-3 inhibition accelerated behavioral deficits, αsyn aggregation, and reductions in TH-positive neurons in the PFF mouse model. Our work here further demonstrates the neuroprotective effects of 14-3-3θ overexpression in multiple brain regions, indicating that this protective mechanism applies broadly to multiple cell types affected by αsyn pathology. Together these data indicate the role of 14-3-3s in the regulation of αsyn pathology and their therapeutic potential as a molecular target for synucleinopathies. Induction of 14-3-3θ may prove to be a viable technique for slowing disease progression.Additional file 1: Figure S1 Verification of sonicated fibril radius by dynamic light scattering (DLS). (a) Representative DLS graph of average sonicated fibril radius shown for WT and 14-3-3θ transgenic mice injected for the 6 mpi time point. After sonication, PFFs were confirmed by Nanodrop for concentration and analyzed by DLS to ensure a mean radius of 20-50 nm for sonicated fibrils. PFFs were reassessed by DLS at the beginning of each day of injections. (b) Representative TEM of αsyn fibrils before sonication. Scale bar = 200 nm. (c) Representative TEM of αsyn fibrils after sonication. Scale bar = 200 nm. Additional file 2: Figure S2 Motor behaviors are not affected by PFF treatment nor by 14-3-3 manipulation in the cortex, amygdala, or nigra. (a–c) Quantification of average velocity (a), distance traveled (b), and percent time in the periphery (c) in the open field test for WT and 14-3-3θ mice injected with αsyn monomer or PFFs at 6 mpi. n = 11–14 per group, *p < 0.05 (Tukey’s multiple comparison test) (Tukey’s multiple comparison test). Error bars represent SEM. (d) Quantification of time to reach the bottom in the pole test for WT and 14-3-3θ mice injected with αsyn monomer or PFFs at 6 mpi. n = 11–14 per group, n.s. (Tukey’s multiple comparison test). Error bars represent SEM. (e) Quantification of latency to fall in the accelerating rotarod test on the second assessment day after 3 days of training for WT and 14-3-3θ mice injected with αsyn monomer or PFFs at 6 mpi. n = 11–14 per group, n.s. (Tukey’s multiple comparison test). Error bars represent SEM.  Quantification of average velocity (f), distance traveled (g), and time in periphery (h) in the open field test for WT and difopein  mice injected with αsyn monomer or PFFs at 6 mpi. n = 14–16 per group, *p < 0.05 (Tukey’s multiple comparison test). Error bars represent SEM. (i–l) Quantification of average velocity (i) and distance traveled (j) in the open field test, time to reach the bottom in the pole test (k), and latency to fall on the wire hang test (l) in AAV-GFP and AAV-14-3-3θ/GFP mice injected with αsyn monomer or PFFs at 6 mpi. n = 12–16 per group. n.s. (Tukey’s multiple comparison test). Error bars represent SEM. (m–p) Quantification of average velocity (m) and distance traveled (n) in the open field test and latency to fall in the accelerating rotarod test  in WT and difopein  mice injected with αsyn monomer or PFFs at 6 mpi. n = 15–17 per group. *p < 0.05 (Tukey’s multiple comparison test). Error bars represent SEM.Additional file 3: Figure S3 αSyn aggregation occurs primarily in neurons at 6 months post injection. (a) Representative images of pS129-αsyn immunostaining in the cortex at 6 mpi in WT and 14-3-3θ mice injected with αsyn monomer into the striatum. Scale bar = 100 μm. (b) Representative images of HA immunostaining in the cortex, SN, and amygdala demonstrates that HA-tagged 14-3-3θ is expressed in cortical and amygdala regions, but not in the SN. Scale bar = 100 μm for cortex and SN; 50 μm for amygdala. (c) pS129-αsyn inclusions associate primarily with NeuN instead of GFAP in PFF-injected WT and 14-3-3θ transgenic mice at 6 mpi. Scale bar = 50 μm.Additional file 4: Figure S4 αSyn aggregation is accelerated in the amygdala in mice expressing difopein in the amygdala. (a) Representative images of eYFP-difopein immunostaining in the amygdala of WT and difopein  mice at 3 mpi. GFP-difopein expression is found only in difopein mice. Scale bar = 100 μm. (b) Representative images of pS129-αsyn immunostaining in the cortex of WT and difopein mice at 3 and 6 mpi. Scale bar = 100 μm. (c) Difopein expression increases inclusion counts in PFF-injected mice at 3 mpi. Quantification of pS129-αsyn positive inclusions at 3 mpi and 6 mpi in the amygdala of PFF-injected WT and difopein mice. n = 11–12 per group at 3 mpi. n = 8–9 at 6 mpi. *p < 0.05 (Student’s t-test). Error bars represent SEM.Additional file 5: Figure S5 pS129-αsyn positive inclusions do not consistently correlate with TH-positive neuronal cell counts. (a) pS129-αsyn positive inclusions and TH-positive cell counts in PFF-injected AAV-14-3-3θ/GFP and AAV-GFP mice at 6 mpi . (b) pS129-αsyn positive inclusions and TH-positive cell counts in PFF-injected difopein and WT mice at 3 mpi . (c) pS129-αsyn positive inclusions and TH-positive cell counts in PFF-injected difopein and WT mice at 6 mpi .Additional file 6: Figure S6 Difopein promotes the reduction of NECAB1-positive neurons in response to PFFs. (a) Representative images of NECAB1-positive immunostaining in the cortex of WT and difopein mice injected with αsyn monomers or PFFs at 6 mpi. Scale bar = 100 μm. (b) pS129-αsyn (red) immunostaining is concentrated in NECAB1-positive (green) IV and V layers of the sensorimotor cortex in a PFF-injected WT mouse. Scale bar = 50 μm. (c) PFF-injected difopein mice have decreased NECAB1 counts in comparison to monomer-injected difopein mice. Quantification of NECAB1-positive neurons in the cortex of WT and difopein mice injected with αsyn monomers or PFFs at 6 mpi. n = 7–8 per group. ****p < 0.0001 (Tukey’s multiple comparison test). Error bars represent SEM.


The zinc aspartate complex, commonly used as a dietary supplement, crystallizes as either the dihydrate or the sesquihydrate. High pressure favours the sesquihydrate, and this has been rationalized by measurements of the compressibility using single-crystal X-ray diffraction up to 4 GPa. 2) complex, a common dietary supplement, preferentially crystallizes as the dihydrate (ZnAsp2·2H2O) from aqueous solution. Under normal conditions the dihydrate easily transforms into the sesquihydrate (ZnAsp2·1.5H2O). The dihydrate crystal structure is triclinic, space group P1, and the sesquihydrate is monoclinic, space group C2/c. However, their structures are closely related and similarly consist of zinc aspartate ribbons parallel to pores accommodating water molecules. These porous structures can breathe water molecules in and out depending on the temperature and air humidity. High pressure above 50 MPa favours the sesquihydrate, as shown by recrystallizations under pressure and compressibility measured by single-crystal X-ray diffraction up to 4 GPa. This preference is explained by the reduced volume of the sesquihydrate and water compressed separately, compared with the dihydrate. The sesquihydrate undergoes an isostructural phase transition when the voids collapse at 0.8 GPa, whereas no phase transitions occur in the dihydrate, because its pores are supported by increased water content.The zinc aspartate (ZnAsp All isochoric high-pressure recrystallizations yielded the sesquihydrate, ZnAsp2·1.5H2O. Its growth process is illustrated in Fig. 2supporting information).We performed high-pressure experiments either by gradually compressing a single crystal in a diamond anvil cell (DAC), or by high-pressure recrystallization and growing single crystals in isothermal and isochoric conditions from aqueous solutions The sesquihydrate could be recovered from the DAC and no visible changes occurred to the samples exposed to air for weeks. On the other hand, the initially crystallized dihydrate powder kept in a thermally closed plastic bag for two months fully transformed into the sesquihydrate. This indicates that the dihydrate is stable only when submerged in water and in highly humid environments.2·1.5H2O and ZnAsp2·2H2O crystals was determined for the samples mounted in the DAC. The gasket was made of 0.2 mm thick Inconel foil and the initial diameter of the spark-eroded hole was 0.45 mm. Glycerine was used as a hydrostatic medium. The pressure in the DAC was calibrated before and after each diffraction measurement by the ruby fluorescence method . The water H atoms were located from difference Fourier maps and then the positions of H2O were refined as rigid units. The Uiso values of the H atoms were constrained to 1.2 times the Ueq of their carrier atoms. Structural drawings were prepared using the program Mercury CSD 3.3  and a LynxEye detector. The samples were gently pressed into a flat round cuvette of about 0.4 cm3 in volume. The cuvette was rotated during the measurements, performed in θ steps of 0.02° and in the θ–2θ mode.Powder X-ray diffraction (XRPD) patterns were recorded using a Bruker AXS D8 Advance diffractometer, equipped with a sealed X-ray tube, a Johansson monochromator selecting Cu 2·2H2O in an N2 atmosphere on a Setsys 1200 Setaram instrument between 293 K and 573 K at a scan speed of 5 K min−1.Thermogravimetric (TG) and differential scanning calorimetry (DSC) analyses were performed for 18.2 mg of ZnAsp3.2·1.5H2O and ZnAsp2·2H2O retain the ambient-pressure monoclinic symmetry of space group C2/c and triclinic symmetry of space group Figs. S2 and S3).The compressed crystals of ZnAsp2 dihydrate and sesquihydrate are strikingly similar. Their Bravais lattices can be transformed into one another through the following matrix M:C lattice of (ZnAsp2·1.5H2O) and the triclinic P lattice of (ZnAsp2·2H2O), respectively. The reverse matrix M−1 transforms the lattice Cm into Pt:P unit cell of ZnAsp2·2H2O at 0.1 MPa can be represented as the pseudo-monoclinic unit cell C, having the following dimensions: a = 16.445 Å, b = 10.804 Å, c = 14.780 Å, α = 85.28°, β = 92.27°, γ = 89.66°, which are similar to the unit cell of ZnAsp2·1.5H2O , with increasing pressure this triclinic structure only hardly, within about 0.5°, changes its distortions from the monoclinic symmetry.Although seemingly very different Table 1, the cryO Table 1. Accordi2·nH2O framework results in its transformation, changing the symmetry of the crystal structure. It is characteristic that the monoclinic symmetry of space group C2/c of ZnAsp2·1.5H2O is reduced to the triclinic space group 2·2H2O after the water content increases. This inverse sorption–symmetry relation is surprising, as according to our survey an increased water content of the pores either increases or preserves the crystal symmetry. For example, the space-group symmetry R3 of anhydrate stepanovite polymorph ST1d increases to space group R3c for stepanovite [Mg(H2O)6][NaFe(C2O4)3]·3H2O; for the polymorph ST2d its anhydrate has space-group symmetry P3 and after hydration the symmetry increases to P3c  polymorphs increase to Cmmm for the dihydrate; and space group 2H3O2)·H2O, increases to P21/c for the tetrahydrate 4(LiC2H3O2)·4H2O . The dihydrate crystals are triclinic .We have established that ZnAspc Table 1. In thes2·1.5H2O and ZnAsp2·2H2O that all water molecules interact through O—H⋯O and N—H⋯O hydrogen bonds, but no water molecules participate in Zn coordination. There are very similar intramolecular N13—H13C⋯O11 and N3—H3A⋯O2 hydrogen bonds in both ZnAsp2·1.5H2O and ZnAsp2·2H2O. Almost all of the hydrogen bonds involve water molecules. The hydrogen bonds to H2O molecules are O1W—H1WB⋯O14, O1W—H1WA⋯O2, O2W—H2WB⋯O12 and N3—H3C⋯O1W. Two other hydrogen bonds are N13—H13B⋯O2 and N3—H3B⋯O13, both present in the ZnAsp2·1.5H2O and ZnAsp2·2H2O structures. The O2W—H2WA⋯O1W bond between two water molecules is formed only in ZnAsp2·2H2O .It is a common feature of both ZnAsp2·2H2O and ZnAsp2·1.5H2O can interconvert one into the other under ambient conditions, each of them can be compressed in glycerine to 4 GPa at least ] and in the volume compression ]. These two phases of ZnAsp2·1.5H2O will be further referred to as phases α and β.It is remarkable that although ZnAspst Fig. 3. Howeverre Fig. 5. A similon Fig. 3. It appe2·1.5H2O structure determined at 0.9 GPa is an average of these phases, as the X-ray diffraction measurement was performed when the sample had partly transformed between phases α and β. The anomalous compression at 0.8 GPa, determined from the unit-cell dimensions, agrees well with the collapse of the pores. Their volume is plotted as a function of pressure in Fig. 62NO3·NO3 transforms between monoclinic phases, both of space-group symmetry P21/c 3(NO3)4·xH2O preserves its orthorhombic space group Ccca at pc = 6 GPa  should give the volume difference corresponding to half of the molecular volume of water [Fig. 6b)]. The comparison of ΔVm with the volume of one H2O molecule in water and ices shows that there is more space in the pores than required for the accommodated water molecules, which explains the reason for the collapse of the pores. The work contribution to the Gibbs free energy at the transition in ZnAsp2·1.5H2O is 3.1 kJ mol−1, compared with the work energy of 4.9 kJ mol−1 performed by the pressure to compress phase α from 0.1 MPa to 0.8 GPa. The volume difference between ZnAsp2·2H2O and ZnAsp2·1.5H2O at 0.1 MPa is consistent with the broad distribution of the volume of solvent water occurring in organic pharmaceuticals  has implications for its use as a pharmaceutical agent. The undesired release of water in tablets results in their degradation, as illustrated in the photograph in Fig. 72·1.5H2O, which is more stable and does not release or absorb water. The sesquihydrate can be obtained either by drying the dihydrate precipitate obtained from aqueous solution, or by performing the crystallization under high-pressure conditions. The high pressure of about 50 MPa required for this purpose is technologically attainable for larger amounts of the sample, although it would require some safety measures. High-pressure crystallization could be more advantageous in terms of the time needed to obtain the required product. Thus the high-pressure crystallization of zinc aspartate can be regarded as a potential candidate for pharmaceutical applications of high pressure  under ambient conditions. We have shown that the precipitate obtained by crystallization from aqueous solution is the dihydrate, ZnAsp2·2H2O. The crystal structures of the sesquihydrate ZnAsp2·1.5H2O and the dihydrate ZnAsp2·2H2O are closely related, despite their different space-group symmetries.We have established that the sesquihydrate ZnAsp2·1.5H2O when it is immersed in water, or leave the pores when the dihydrate ZnAsp2·2H2O is exposed to the air. This transformation of the dihydrate into the sesquihydrate is a slow process, taking days or weeks depending on the volume of the sample, its container, the size of the crystal grains, the humidity of the air, ventilation, temperature and other relevant parameters. Thus it is recommended to check whether the transformation into the sesquihydrate has been completed before further processing, for example granulating or tabletting.The most eminent feature of these structures is that they form frameworks containing channel pores that are occupied by water molecules. The water molecules can enter the pores of the sesquihydrate ZnAspThe sesquihydrate can also be obtained directly by high-pressure crystallization. We found that an applied pressure as low as 50 MPa favours crystallization of the sesquihydrate from aqueous solution at 298 K.2·1.5H2O illustrates the role of directional interactions in stabilizing porous structures. It can be postulated that the sesquihydrate is favoured by high pressure because it increases the close packing of molecules in the structure and destabilizes the directional interactions, like hydrogen bonds, which bind the water molecules into the pores. However, it is apparent from other reverse effects on other hydrates that the pressure effects are quite complex and depend on specific structural features.The subtle isostructural phase transition in ZnAsp2 hydrates never resulted in the separation of enantiomers. This negative result is consistent with the effect of pressure on other racemic mixtures of enantiomers reported so far  global, Zn-aspartate_sesquihydrate_0_001GPa, Zn-aspartate_sesquihydrate_0_05GPa, Zn-aspartate_sesquihydrate_0_29GPa, Zn-aspartate_sesquihydrate_0_49GPa, Zn-aspartate_sesquihydrate_0_79GPa, Zn-aspartate_sesquihydrate_0_85GPa, Zn-aspartate_sesquihydrate_1_15GPa, Zn-aspartate_sesquihydrate_1_44GPa, Zn-aspartate_sesquihydrate_1_62GPa, Zn-aspartate_sesquihydrate_1_82GPa, Zn-aspartate_sesquihydrate_2_15GPa, Zn-aspartate_sesquihydrate_2_49GPa, Zn-aspartate_sesquihydrate_2_91GPa, Zn-aspartate_sesquihydrate_3_54GPa, Zn-aspartate_sesquihydrate_3_92GPa, Zn-aspartate_dihydrate_0_001GPa, Zn-aspartate_dihydrate_0_05GPa, Zn-aspartate_dihydrate_0_15GPa, Zn-aspartate_dihydrate_0_40GPa, Zn-aspartate_dihydrate_0_70GPa, Zn-aspartate_dihydrate_1_02GPa, Zn-aspartate_dihydrate_1_54GPa, Zn-aspartate_dihydrate_2_53GPa, Zn-aspartate_dihydrate_3_05GPa, Zn-aspartate_dihydrate_3_53GPa, Zn-aspartate_dihydrate_4_02GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_0_001GPasup2.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_0_001GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_0_05GPasup3.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_0_05GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_0_29GPasup4.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_0_29GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_0_49GPasup5.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_0_49GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_0_79GPasup6.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_0_79GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_0_85GPasup7.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_0_85GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_1_15GPasup8.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_1_15GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_1_44GPasup9.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_1_44GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_1_62GPasup10.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_1_62GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_1_82GPasup11.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_1_82GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_2_15GPasup12.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_2_15GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_2_49GPasup13.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_2_49GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_2_91GPasup14.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_2_91GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_3_54GPasup15.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_3_54GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_sesquihydrate_3_92GPasup16.hklStructure factors: contains datablock(s) zinc-aspartate_sesquihydrate_3_92GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_0_001GPasup17.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_0_001GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_0_05GPasup18.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_0_05GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_0_15GPasup19.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_0_15GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_0_40GPasup20.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_0_40GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_0_70GPasup21.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_0_70GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_1_02GPasup22.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_1_02GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_1_54GPasup23.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_1_54GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_2_53GPasup24.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_2_53GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_3_05GPasup25.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_3_05GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_3_53GPasup26.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_3_53GPa. DOI: 10.1107/S2052520620009348/lo5070zinc-aspartate_dihydrate_4_02GPasup27.hklStructure factors: contains datablock(s) zinc-aspartate_dihydrate_4_02GPa. DOI: 10.1107/S2052520620009348/lo5070sup28.pdfAdditional tables and figures. DOI: 1972145, 1972146, 1972147, 1972148, 1972149, 1972150, 1972151, 1972152, 1972153, 1972154, 1972155, 1972156, 1972157, 1972158, 1972159, 1972160, 1972161, 1972162, 1972163, 1972164, 1972165, 1972166, 1972167, 1972168, 1972169, 1972170CCDC references:


Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a “chaperone” to promote 3′ to 5′ filament growth via highly dynamic engagement with 5′ filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The   •C. elegans BRCA2 and Rad51 paralogs synergistically promote Rad51 filament assembly•Rad51 paralogs dynamically engage with the 5′ Rad51 filament ends•Rad51 paralogs stimulate Rad51 filament growth in a 3′–5′ direction•in vivoRetention of Rad51 paralogs at filament ends hinders growth and blocks HR  C. elegans Rad51 filament assembly in the presence of recombination mediators. BRCA2 primarily enhances Rad51 nucleation on ssDNA, whereas Rad51 paralogs dynamically engage with 5′ Rad51 filament ends and stimulate filament growth in a 3′–5′ direction.Belan et al. exploit single-molecule approaches to reveal the mechanism of  Homologous recombination (HR) is a largely error-free mechanism of DNA double-strand break (DSB) repair. DSBs can arise spontaneously because of replication fork stalling or collapse or after exposure to DNA-damaging agents, such as ionizing radiation. HR repair is also essential to produce inter-homolog crossovers necessary for correct chromosome segregation at the first meiotic division .HR is a complex process comprising several conserved steps. First, broken double-stranded DNA (dsDNA) ends are nucleolytically processed, yielding 2–4 kb of single-stranded DNA (ssDNA) coated by replication protein A (RPA). RPA is displaced by Rad51, a eukaryotic recombinase that forms helical nucleoprotein filaments with single-stranded DNA (ssDNA). Within the filament, DNA is extended 1.5-fold over the dsDNA contour length, with a stoichiometry of 3 nt per RAD51 monomer and six RAD51 monomers per helical turn . Rad51 fGiven the lower affinity of Rad51 for ssDNA, compared with RPA, mediator proteins that promote RPA displacement are crucial for efficient HR. In higher eukaryotes, BRCA2 and Rad51 paralogs  are among the most critical mediator proteins. Loss of BRCA2 or individual RAD51 paralogs results in sensitivity to DSB-inducing agents, loss of Rad51 foci at sites of DSBs and defective HR repair . MutatioBiochemical studies have shown that sub-stoichiometric amounts of full-length human BRCA2 promote nucleation of RAD51 on ssDNA and enhance its strand-exchange activity in bulk assays . BRCA2 aE. coli RecA recombinase have revealed that nucleoprotein filaments rapidly form in the presence of bacterial single-stranded binding protein (SSB) by a two-step mechanism: rate-limiting nucleation, followed by rapid, bi-directional filament growth with a 2-fold kinetic preference for the 5′→3′ direction along an ssDNA backbone , a recombination mediator and ortholog of the breast and ovarian cancer tumor-suppressor protein, BRCA2 , we employed a “dipping protocol”  assessed after a single round of dipping in the presence of ATP displays power dependence with respect to RAD-51f concentration, according to kobs = J[RAD-51]n, in which J is a rate constant and n is the number of protomers in a minimal nucleation unit. In agreement with studies of bacterial RecA (n = 1.5 in ATP and n = 2.2 in ATP-γ-S) (C. elegans RAD-51)     and huma RAD-51) C and S4Ds result B and 1C,s result D. Additif clusters over time in the presence of RFS-1/RIP-1(A647) and ATP  molecules revealed that they bind to RAD-51ll times E and 4F.clusters G is consf clusters are dimeric  stabilizing RAD-51f on ssDNA, we observe more-frequent growth in the number of RAD-51f protomers in individual clusters  association with RPA-eGFP-coated ssDNA in the presence of RAD-51 and ATP A and 5B.We have established that RAD-51 filaments grow preferentially in the 3′ to 5′ direction in the presence of RFS-1/RIP-1. We have also shown that RFS-1/RIP-1 shuts down dissociation of RAD-51 clusters on ssDNA leading to increased dwell times and growth frequencies. This prompted us to investigate whether labeled RFS-1/RIP-1 directly binds to 5′ filament ends using gapped DNA substrates with defined polarity. To directly visualize RFS-1/RIP-1 at filament ends, we incubated RAD-51 filaments with 100 nM RFS-1/RIP-1(A647) C, which in vitro  deletion strain, rfs-1 K56A and rfs-1 K56R mutant strains display a modest increase in the levels of embryonic lethality compared with the N2 (WT) strain without significant brood-size reduction , camptothecin (CPT), and nitrogen mustard (HN2), although to a lesser extent than rfs-1-null animals are. Sensitivity to hydroxyurea (HU) is not observed in the rfs-1 K56A and rfs-1 K56R mutant strains  might further stimulate filament growth enhancement by BRCA2, particularly given that both PALB2 and DSS1 enhance the stimulatory effect of BRCA2 in bulk DNA strand exchange assays . Rad51 pBacterial RecA filaments were previously shown to grow bi-directionally with a 2-fold kinetic preference for the 5′→3′ direction . Using aPrevious work using bulk EM imaging postulated that Rad51 paralogs stabilize Rad51 filaments by stably intercalating into them . In contWe further establish that this rapid turnover is dependent on ATPase activity of RFS-1 and demonstrate that RFS-1 Walker box mutations or blocking ATP hydrolysis stabilizes RAD-51 on ssDNA, but failure to disengage RFS-1/RIP-1 from 5′ filament ends hinders further filament growth. Taken together, our results suggest that RFS-1/RIP-1 acts as RAD-51 filament chaperone by recognizing the nascent 5′ RAD-51 filament ends, transiently binding them to prevent filament disassembly, and dissociating to permit further Rad51 protomer addition and filament growth. Iterative cycles allow for efficient filament extension similar to how classical molecular chaperones mediate unfolded protein recognition, folding, and release cycles.rfs-1-null and Walker A box K56A/R mutant strains. Loss of RFS-1 in the null strain confers marked sensitivity to DNA damaging agents accompanied by the loss of RAD-51 foci. Strikingly, in rfs-1 Walker A box K56A/R knockin strains, DNA damage sensitivity is also observed, but RAD-51 foci extensively accumulate, indicative of stabilized, but HR-incompetent, RAD-51 complexes. Extensive RAD-51 foci observed in rfs-1 K56A/R background may result from continued resection and RAD-51 loading by BRC-2 as perturbations of downstream HR are known to increase resection tract length  means that it is possible multiple RAD-51 nucleation events can occur within a single pixel. This would influence overall nucleation and growth rates as discussed. Given our reductionist approach, other components of RAD-51 assembly reaction have not been studies, such as accessory RAD-51-binding factors or posttranslational modifications, which could also influence the overall results. Finally, findings with nematode proteins might not be fully applicable to the human system, where the recombinase-ssDNA assembly mechanism might differ to a certain extent.simon.boulton@crick.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Simon J. Boulton (simon.boulton@crick.ac.uk).Plasmids, recombinant proteins, DNA substrates and newly generated nematode strains are available without restriction upon requests, which should be directed to the Lead Contact, Simon J Boulton .Custom-written data analysis scripts are available upon request from David S. Rueda (E. coli strain (genotype: fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) was transformed with protein expression plasmids (KEY RESOURCE TABLE) and grown in Luria Broth at 37°C in the presence of 50 μg/ml kanamycin. BL21(DE3) E. coli strain  [dcm] ΔhsdS; λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5) was transformed with protein expression plasmids (KEY RESOURCE TABLE) and grown in Luria Broth at 37°C in the presence of 50 μg/ml kanamycin and induced by 1 mM IPTG at 30°C for 4 h. OP50 E. coli strain was grown at 37°C in Luria Broth, spread on NGM agar plates and kept at 20°C.DH5α S. cerevisiae yJF1 (W303-1a pep4::KanMx4 bar1::Hph-NT1 ade2-1 ura3-1 his3-11 trp1-1 leu2-3) strain grown in YP + 2% raffinose and induced by the addition of 2% galactose. Nematode strains were propagated on NG agar plates seeded with E. coli (OP50) at 20-25°C.Nematode RFS-1/RIP-1-3xFLAG complex and its variants were overexpressed from integrated pRS303 plasmid in S. cerevisiae yJF1 strain (genotype: W303-1a pep4::KanMx4 bar1::Hph-NT1 ade2-1 ura3-1 his3-11 trp1-1 leu2-3) strain had pRS303 plasmid variants (KEY RESOURCE TABLE) stably integrated into the genome and was grown at 30°C in YP media supplemented with 2% raffinose. Protein induction was done by the addition of 2% galactose for 4 h at 30°C. S. cerevisiae MaV103 strain  were transformed with Y2H protein expression plasmids and grown at 30°C at YPD agar plates or selection plates .C. elegans strains used in the study: ATG563 (genotype: rfs-1(fq127 [K56R]) III/hT2 ), ATG567 (genotype: rfs-1(fq130 [K56A]) III/hT2 ), ATG600 (genotype: brc-2(fq140[HA::AID::brc-2]) III; ieSi38  IV), ATG608 (genotype: brc-2 (fq140 [HA::AID::brc-2]) rfs-1 (fq130 [K56A]) III;; ieSi38  IV), RB1279 (genotype: rfs-1(ok1372) III) and N2 Bristol were maintained at 20°C on NG agar plates seeded with E. coli (OP50). Generation of new C. elegans strains is described in detail in Individual C. elegans RPA remains unknown  was expressed in BL21(DE3) E. coli strain at 17°C overnight using 0.1 mM IPTG for the induction of protein expression. Cells were lysed in Lysis Buffer , cat no. 11873580001, Roche). After sonication and centrifugation at 20,000 rpm for 1h, clarified lysate was applied to Ni-NTA  resin for 1.5h, washed with Lysis buffer and Lysis buffer containing 20 mM imidazole. Proteins were eluted using Elution buffer 500 . Sample was then directly applied to amylose resin and allowed to be bound for 1h, amylose beads were washed with Wash buffer 300 . Protein was eluted using Elution buffer 300  and diluted two times with Elution buffer lacking KCl and maltose). Sample was then loaded onto pre-equilibrated HiTrap SP column, column was washed with 10 column volumes of Buffer A  and eluted using linear salt gradient (0%–80%) of Buffer B . Fractions containing BRC-2 were pooled, concentrated, frozen, and subsequently checked for purity using SDS-PAGE. Ability of his6-MBP-BRC-2 to stimulate RAD-51 in DNA strand exchange in sub-stoichiometric amounts was confirmed using previously established protocol , 200 mM KCl, 0.1 mM DTT, and 25% glycerol). Dyes were diluted in dry DMSO to 50 mM. Dyes and protein were mixed to final concentration of 50 μM protein and 500 μM FAM-SE or 150 μM Cy3/Cy5-NHS. Incubation on rotary shaker at 4°C followed for 2 h 45 min (FAM-SE) or 2h . Reaction was terminated by the addition of Tris–HCl (pH 7.5) to a final concentration of 50 mM. Proteins were then buffer exchanged at least twice into storage buffer . Protein concentration was estimated by Coomasie staining and dye concentration was measured spectrophotometrically. Presence of minimum free dye concentration was assessed using SDS-PAGE on labeled proteins. Protein to dye concentration ratio was consistently 0.8-1.0.RAD-51 was labeled using amine-reactive FAM, Cy5 and Cy3 NHS-esters as described previously for RecA with modifications . Briefly2, 1 mM DTT. After overnight incubation at 4 °C, the labeled protein complex was purified away from Sfp and free dye using Zeba column gel filtration system . Proteins were stored in 20 mM Tris-acetate (pH 8.0), 100 mM potassium acetate, 10% glycerol, 1 mM EDTA, 0.5 mM DTT and subjected to SDS-PAGE and the fluorescent gel was scanned with a Typhoon9500 Scanner.For RFS-1/RIP-1 labeling we genetically fused ybbr tag (DSLEFIASKLA) on the C terminus of RIP-1 downstream of 3xFLAG tag, separated by GGGSGGG linker. Proteins were expressed and purified using a previously established protocol . The lab2) supplemented with 1mM ATP, oxygen scavenging system  and 0.2 mg/ml BSA. Proteins were flowed into the system in 1xNTM buffer  supplemented with 1 mM ATP, oxygen scavenging system  and 0.2 mg/ml BSA. When high protein concentrations were used (≥500 nM), ATP-regeneration system consisting of 20 mM phospho-creatine and 20 μg/mL creatine kinase was also added into the reaction.Experiments were performed using commercially available C-trap (LUMICKS) setup. Protein channels of the microfluidics chip were first passivated with BSA (0.1% w/v in PBS) and Pluronics F128 (0.5% w/v in PBS), minimum 500 μL of both flowed through prior to use. Biotinylated ssDNA precursor was prepared as described previously . To gene2+ and ADP-aluminum fluoride-Mg2+ were performed in addition to ATP-γ-S. However, no RFS-1/RIP-1(A647) binding to RAD-51f clusters was observed under these conditions. BRC-2 was also assessed on bare ssDNA in the ‘dipping assay’, however, we observed formation of extremely bright aggregated GFP-BRC-2-ssDNA clusters on ssDNA containing numerous (> 20) molecules of BRC-2, which was not the case under physiological conditions with RPA. For confocal imaging, three excitation wavelengths were used, 488 nm for eGFP and 6-FAM, 532 nm for Cy3 and 638 nm for Cy5, with emission detected in three channels with blue filter 512/25 nm, green filter 585/75 nm and red filter 640 LP. Imaging conditions for ‘dipping assay’: 15% laser power, 0.1ms/pixel dwell-time, 100 nm pixel size. Imaging conditions for ‘RPA-eGFP displacement assay’: 2% blue laser power, 5% red laser power, 0.1ms/pixel dwell-time, 100 nm pixel size, 1.5 s inter-frame wait time.For ‘dipping assays’ performed with different cofactors, controls using AMP-PNP-Mgf clusters in minimal imaging area to obtain sufficiently high bleaching time resolution. Scans were sectioned and stacked in Fiji using a custom-written script. Maximum likelihood estimation was used to determine each of the photobleaching steps within a maximum intensity/frame n. trace as previously described ). Worm-like chain (WLC) model for λ dsDNA was used as a reference for force-extension curve comparison. Custom-written WLC fitting script was used to calculate contour length and subsequently gapped length of gapped DNA substrates. Growth rates in real-time experiments as well as dwell-times and binding frequencies were estimated in Fiji. Nucleation frequencies were plotted as a function of RAD-51f concentration and fitted with power-law in GraphPad Prism 7. Dwell-times of RAD-51 clusters were binned into appropriate dwell-time categories cumulative survival analysis was performed using Igor 8.0. Mann-Whitney test was used to assess statistical significance of the data where appropriate.15% blue laser power was used to bleach RAD-51escribed . The ste2 and 2 mM ATP, and incubated for 10 min, before crosslinking with 0.25% glutaraldehyde for 10 min, all at 25°C. Reactions were resolved on 1% agarose gels in 1X TBE . Gels were dried and imaged by autoradiography or using a storage phosphor screen (Amersham Biosciences) and Typhoon9500 and quantified using Fiji. For fluorescence experiments using RAD-51f and/or labeled RFS-1/RIP-1 complex, proteins were incubated with 20 nM 49-mer oligonucleotide AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT in 35 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 2 mM MgCl2, 2 mM ATP and incubated for 10 min at 25°C followed by resolution on 0.8% agarose gel in 1X TAE . Gels were dried ad imaged uing Typhoon9500 and appropriate filter settings. % of DNA binding was assessed using Fiji.Proteins were diluted from concentrated stocks into Storage Buffer, which was also used in no protein controls. For native polyacrylamide gels, proteins were mixed with a master mix (containing 60 nM (nucleotides) 5′-[32P]-labeled 60-mer oligonucleotide (ACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC), 20 mM Tris-HCl (pH 7.5), 8% glycerol, 1 mM DTT, 50 mM sodium acetate, 2 mM MgClf were diluted from concentrated stocks into T Buffer (25 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA (pH 8.0), 100 mM KCl), which were also used in no protein controls. Proteins were mixed with 30 nM Cy5-labeled 90-mer ssDNA in 35 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 2 mM MgCl2 and 2 mM ATP and incubated for 10 min at 25°C followed by addition of 0.54 μg pBS(-) dsDNA plasmid for further 15 min incubation at 25°C. Reactions were terminated by SDS-PK treatment for 10 min at 37°C. 90V/35 min resolution using 1xTAE, 0.8% agarose gel electrophoresis followed. Gels were scanned using Typhoon9500 with appropriate filter settings.RAD-51 and RAD-512, and stored at stock concentration 200 nM (moles). Proteins were diluted from concentrated stocks into T Buffer (25 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA (pH 8.0), 50 mM KCl), which was also used in no protein controls. Proteins were mixed with 5.6 nM (moles) 150-mer oligonucleotide (TCTTATTTATGTCTCTTTTATTTCATTTCCTATATTTATTCCTATTATGTTTTATTCATTTACTTATTCTTTATGTTCATTTTTTATATCCTTTACTTTATTTTCTCTGTTTATTCATTTACTTATTTTGTATTATCCTTATCTTATTTA), 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 100 μg/ml of BSA, 2 mM ATP, 4 mM CaCl2 in 12.5 μL reaction volume at 25°C for 10 min. 0.5 μL dsDNA stock and 0.5 μL 0.1 M spermidine were then added incubated for 1:30 h. The samples were deproteinized with 0.1% SDS and 12.5 μg proteinase K at 37°C and resolved in 10% polyacrylamide gels in 1X TBE . Gels were imaged on a Typhoon9500 and quantified using Fiji.40-mer dsDNA was prepared by annealing 5′-fluorescein-labeled 40-mer oligonucleotide (TAATACAAAATAAGTAAATGAATAAACAGAGAAAATAAAG) to the complementary unlabelled 40-mer oligonucleotide (CTTTATTTTCTCTGTTTATTCATTTACTTATTTTGTATTA) in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 2 mM ATP for 5 min °C. For negative staining, Quantifoil R2/2, 2 nm carbon, 400 Cu mesh grids were glow discharged for 30 s at 25 mA with a K100X glow discharger (EMS), 4 μL of sample was added to the grid left for 1 min. Excess sample was blotted away leaving a thin film. Then the grid was dipped into buffer solution twice and dipped twice into 2% uranyl acetate solution, blotting in between. Negative stain EM data were acquired on Tecnai Spirit TEM operated at 120 kV, equipped with a n FEI Eagle CCD camera.RAD-51 and RFS-1/RIP-1 in indicated concentrations were incubated with 250 nM (in nucleotides) 150-mer poly(dT) ssDNA in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCldpy-10 was used as a co-injection marker to select progeny carrying Cas9-induced edits. The following sequences were used to generate crRNAs (IDT): rfs-1 K56 mutants: TTTAGGAGTTGGTAAAACAC; HA::AID::brc-2: TTTTTAGATGAGTCACCCAT; dpy-10: GCTACCATAGGCACCACGAG. The repair templates used (Table S1) were ordered as single-stranded DNA oligos at 4 nmol (IDT).Genome editing by CRISPR-Cas9 was performed using preassembled Cas9-sgRNA complexes  and single-stranded DNA oligos (used as repair templates) as described before . dpy-10 dpy-10 crRNA:trRNA duplex (100 μM), and 2.95 μl of Cas9 nuclease V3  and incubating the mix at room temperature for 5 min. The final injection mix was prepared by adding 0.6 μl of each ssDNA repair template from a 100 μM stock and 0.5 μl of dpy-10 repair template (10 μM stock) to 5.15 μl of the Cas9/crRNA/trRNA complex, the mix was completed with H2O to obtain a final volume of 10 μl. The injection mix was directly injected into the gonads of young adult worms. Following injection, worms were placed onto individual NG agar plates seeded with E. coli (OP50) and incubated at 25°C for three days. Roller and dumpy worms, caused by Cas9-dependent editing of the dpy-10 gene, were picked individually to plates and allowed to produce progeny that was screened by PCR for the presence of the desired edit.crRNAs and trRNA were reconstituted with nuclease-free duplex buffer to 200 μM and mixed in equal volumes to generate crRNA:trRNA duplex at 100 μM. Cas9/crRNA/trRNA complexes were generated by adding 2 μl of crRNA:trRNA duplex (100 μM) of the target gene, 0.2 μl of ieSi38 transgene, expressing TIR-1-mRuby under the sun-1 promoter  seeded with E. coli (OP50) and allowed to lay eggs for 2 h. Embryos were cultured on the auxin-containing plates for three days before young adults were picked and processed for immunostaining.Auxin-mediated degradation of BRC-2 in the germline was performed by creating a strain homozygous for the promoter , and forExposure of worms to indicated doses of Cis-Diammineplatinum (II) dichloride , Hydroxyurea , bis(2-chloroethyl)methylamine , and (S)-(+)-Camptothecin , was performed by placing worms on NG agar plates containing the desired amount of each genotoxic agent. Randomly picked young adult animals were placed on MYOB plates containing 200 μM CDDP, 500 nM CPT, 60 μM HN2 cisplatin or control plates. 3-5 worms were plated on each plate. Worms were moved every 24 h to new drug-containing plates. Embryonic survival of progeny was then determined by determining the number of hatched eggs  on the 0–24, 24–48, and 48–72 h plates. For HU treatment, worms were plated on plates containing indicated concentration of HU, for indicated period of time. Animals were transferred to HU–free plates and allowed to recover for 3 h. Worms were then allowed to lay eggs for 4 h. Dead eggs were counted 24 h after removing the parent animals.2, 2 mM CaCl2, 2 mM MgCl2, 5 mM HEPES at pH 7.4) and fixed in 1% paraformaldehyde containing 0.1% Tween for 5 min. Slides were frozen in liquid nitrogen, then immersed for 1 min in methanol at –20°C and transferred to PBST . After washing the slides three times in PBST for 5 min, they were blocked in PBST 0.5% BSA for 30 min before incubating then overnight at room temperature with PBST containing anti-RAD-51 antibodies (a kind gift from A. Gartner) diluted 1:500 were incubated overnight at room temperature. Following three washes of 10 min each in PBST, slides were incubated with secondary antibodies  for two h in the dark. Following three washes of 10 min each in PBST, slides were counterstained with DAPI, washed in PBST for 1 h and mounted using Vectashield. All images were acquired as stacks of optical sections with an interval of 0.2 μm using a Delta Vision deconvolution system equipped with an Olympus 1X70 microscope using 100xlens. Images were subjected to deconvolution using SoftWoRx 3.0 (Applied Precision).Randomly picked gravid adult hermaphrodites were treated with cisplatin  for 19 h and camptothecin  for 18 h in liquid culture Ionizing irradiation and UV-C (254 nm) treatment were performed on seeded plates. UV-C treatment of worms was performed on seeded plates using BLX-254 instrument. After treatment, animals were transferred to fresh seeded plates and allowed to recover . Worms were washed twice in PBS, transferred to poly-L-lysine coated slides and germlines dissected. Germlines from young adults hermaphrodites were dissected in egg buffer ). Worm-like chain (WLC) model for λ dsDNA was used as a reference for force-extension curve comparison. Custom-written WLC fitting script was used to calculate contour length and subsequently gapped length of gapped DNA substrates. Growth rates in real-time experiments as well as dwell-times and binding frequencies were estimated in Fiji. Nucleation frequencies were plotted as a function of RAD-51f concentration and fitted with power-law in GraphPad Prism 7. Dwell-times of RAD-51 clusters were binned into appropriate dwell-time categories cumulative survival analysis was performed using Igor 8.0. Mann-Whitney or other statistical test was used to assess statistical significance of the data where appropriate – sample size and statistical significance are indicated in the figures.Scans were sectioned and stacked in Fiji using a custom-written script. Maximum likelihood estimation was used to determine each of the photobleaching steps within a maximum intensity/frame n. trace as previously described . The ste


The CdII atom has a distorted octahedral coordination environment defined by two nitrogen atoms from one H2bmtz ligand, three oxygen atoms from three different sulfate anions, and one oxygen atom from a coordinating water molecule. The 1,4-dihydro-1,2,4,5-tetrazine ring of the H2bmtz ligand is located about an inversion center, with the NH group being equally disordered over two sites. The sulfate anion acts as a μ3-bridging ligand to connect three CdII atoms, resulting in the formation of [Cd(SO4)(H2O)] sheets propagating parallel to the bc plane. Adjacent sheets are interconnected across the H2bmtz ligands, which coordinate the CdII atoms in a bis-bidentate coordination mode, to form a three-dimensional framework structure. The framework is further stabilized by classical O—H⋯O hydrogen bonds involving the coordinating water molecules and the sulfate groups, and by N—H⋯O hydrogen bonds between the disordered tetrazine NH groups and sulfate oxygen atom, along with C—H⋯π and π–π stacking [centroid-to-centroid separation = 3.5954 (15) Å] interactions between parallel pyrimidine rings of the H2bmtz ligand.The polymeric title compound, poly[aquahemi[μ The structural organization of CPs can result in chains, sheets or three-dimensional frameworks (H2bmtz)0.5(H2O)]n (I) was isolated under hydrothermal conditions. The crystal structure and supramolecular interactions of (I) are reported herein.In this work, to explore the synthesis of novel CPs using 3,6-bis(pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazine, CII cation, one-half of the H2bmtz ligand, one sulfate anion and one coordinating water molecule. The 1,4-dihydro-1,2,4,5-tetrazine ring of the H2bmtz ligand is located about an inversion centre, with the NH group (N4) being equally disordered over two sites. As shown in Fig. 1II atom exhibits a distorted octahedral [CdN2O4] coordination environment with two nitrogen atoms from the H2bmtz ligand, three oxygen atoms from three different sulfate anions and one oxygen atom from the coordinating water molecule. The bond angles around the central CdII atom range from 69.69 (5) to 168.46 (5)°. The Cd—O and Cd—N bond lengths fall in the range of 2.2321 (12)–2.3790 (13) Å, which is comparable with those of reported cadmium(II) sulfate compounds containing additional nitrogen donor ligands such as [Cd2(SO4)2(C16H12N6)2(H2O)2]·4H2O 2(S2O8)(C15H11N2)2(H2O)2]·7H2O (H2O)6(SO4)2]·H2O  with the central six-membered ring of the 1,4-dihydro-1,2,4,5-tetrazine moiety in a twist-boat conformation; the C5—N3—N4Ai—C5i torsion angle is 36.4 (4)° . The sulfate anion acts as a μ3-bridging ligand to connect three CdII atoms to form a sheet-like structure of [Cd(SO4)(H2O)] units, propagating parallel to the bc plane, Fig. 22bmtz ligands, which exhibit a bis-bidentate coordination mode, giving rise to a three-dimensional framework structure, Fig. 3The asymmetric unit of the title compound consists of one CdCg = 3.34 (2) Å; Cg is the centroid of the pyrimidine ring] and π–π stacking  interactions between the pyrimidine rings of the H2bmtz ligand are also observed, Fig. 4In the crystal, classical O—H⋯O hydrogen bonds exist between the coordinating water molecules and the sulfate groups, and N—H⋯O hydrogen bonds involving the disordered tetrazine NH group and sulfate oxygen atoms. In this way, rings with et al., 20162bmtz complexes with transition metals ions, viz. with CuI  and the H2bmtz ligand  in water (5 ml) was added into a 15 ml Teflon-lined reactor, stirred at room temperature for 10 min, sealed in a stainless steel autoclave and placed in an oven. The mixture was heated to 383 K under autogenous pressure for 48 h, and then cooled down to room temperature. After filtration, brown block-shaped crystals were obtained in 80% yield (33.4 mg) based on the cadmium(II) source.All reagents were of analytical grade and were used as received without further purification. The ligand 3,6-bis(pyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazine was synthesized according to a literature method  were used for its refinement. All hydrogen atoms were found in difference-Fourier maps. H atoms attached to C atoms were refined in the riding-model approximation with C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C). The H atoms bound to O or N atoms were refined with distance restraints of O—H = 0.84 ± 0.01 Å and N—H = 0.86 ± 0.01 Å and with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N), respectively.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020006830/wm5546sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020006830/wm5546Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020006830/wm5546Isup3.cdxSupporting information file. DOI: 2004950CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-dibenzooxaborinin-6-ide anion with two different cations, namely, potassium 6,6-difluoro-6H-dibenzooxaborinin-6-ide featuring a polymeric structure, and bis(tetraphenylphosphonium) bis acetonitrile trisolvate, which is composed of discrete cations, anions and acetonitrile solvent molecules linked by C—H⋯O, C—H⋯N and C—H⋯F hydrogen bonds.The crystal structures are reported of the 6,6-difluoro-6 H-dibenzooxaborinin-6-ide  anion with two different cations, namely, potassium 6,6-difluoro-6H-dibenzooxaborinin-6-ide, K+·C12H8BF2O−, (II), featuring a polymeric structure, and bis(tetraphenylphosphonium) bis acetonitrile trisolvate, 2C24H20P+·2C12H8BF2O−·3CH3CN, (III), which is composed of discrete cations, anions and acetonitrile solvent molecules linked by C—H⋯O, C—H⋯N and C—H⋯F hydrogen bonds. There are only minor differences in the geometrical parameters of the anions in these structures.The crystal structures are reported of the 6,6-difluoro-6 Recently, we have prepared various 9-substituted oxaboraphenanthrene derivatives and investigated their stability and luminescence behavior   in the presence of KHF2 in methanol, a clean and quantitative OH/F exchange was established and 9,9-difluorido-10,9-oxaboraphenanthrene potassium (II) was obtained  with Ph4PBr gave the 9,9-difluorido-10,9-oxaboraphenanthrene salt as an acetonitrile solvate (III). The crystal structures of (II) and (III) are now described.Because certain borinic acids are readily fluorinated with KHFI) Fig. 1 could beed Fig. 1. SubsequII) has an almost planar skeleton   is 11.09 (9) Å.The anion in (s) Fig. 2 and the III) consists of two C12H8BF2O− anions . The bond lengths involving B and O  show some slight variations between (II) and (III). In (II), the B—F bonds are longer than in (III), which is presumably due to the F⋯K contacts.The asymmetric unit of  features a polymeric network forming layers lying parallel to (001)   Fig. 7, the comI) Fig. 7 into a tet al., 2016H-dibenzooxaborinine anion. When the F ligands in the query are changed to any ligand, three structures are found, namely (tetrahydrofuran)(tetramethylethylenediamine)lithium lithium benzene solvate bis(tetrahydrofuran)lithium tris(tetrahydrofuran)lithium  and (III).There are no structures in the Cambridge Structural Database  and (III), the B—O bond is significantly longer than in (II) and (III). It is notable that the oxaboraphenanthrene moieties in CADVIN and TUZTAL are far more distorted from planarity than in (II) and (III): the dihedral angles between the aromatic rings are 16.9° in CADVIN and 19.1° in TUZTAL. These dihedral angles are 11.09 (9)° in (II) and 4.53 (7) and 9.68 (8)° in (III).The bond lengths involving B and O in these structures are: CADVIN: B—C = 1.589, B—O = 1.534, C—O = 1.360 Å; TUZTAL: B—C = 1.611, B—O = 1.543, C—O = 1.358 Å. Whereas the B—C and the O—C bonds are in the same range as in I)  in methanol (70 ml) a solution of KHF2  in methanol (30 ml) and water (30 ml) was added at room temperature and the resulting reacting mixture was stirred for 12 h. After removal of all volatiles in vacuo, the residue was extracted into acetonitrile (30 ml). The colorless suspension was filtered. All volatiles were removed in vacuo. The product was obtained as a colorless powder in a yield of 66% . Slow evaporation of a saturated acetonitrile solution of (II) over Granopent led to colorless plates, which were suitable for an investigation by X-ray crystallography.To a solution of 9-hydroxooxaboraphenanthrene II)  in acetonitrile (25 ml) Ph4PBr  was added at room temperature and the resulting reacting mixture was stirred for 12 h. The colorless suspension was filtered. After removal of all volatiles, the product was obtained as a colorless powder in a yield of 92% . Slow evaporation of a saturated acetonitrile solution of (III) over Granopent led to colorless blocks, which were suitable for an investigation by X-ray crystallography.To a solution of (Uiso(H) = 1.2Ueq(C) or with Cmethyl—H = 0.98 Å and with Uiso(H) = 1.5Ueq(C). The methyl groups were allowed to rotate but not to tip. In (III), the C≡N group of one acetonitrile molecule is disordered over two sets of sites with a site occupation factor of 0.545 (5) for the major disorder component.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020015066/hb7952sup1.cifCrystal structure: contains datablock(s) II, III, global. DOI: 10.1107/S2056989020015066/hb7952IIsup2.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020015066/hb7952IIIsup3.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989020015066/hb7952IIsup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015066/hb7952IIIsup5.cmlSupporting information file. DOI: 2043934, 2043933CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-019-40012-y, published online 05 March 2019Correction to: The Supplementary Figure file that accompanies this Article contains an error in Supplementary Figure S1-1, whereby it was incorrectly extended to 1300s. The correct Figure S1-1 appears below as Figure Additionally, the Supplementary Information file that accompanies this Article contains errors in Supplementary Table S8-2.The row entitled “Initial concentration (d02)” under the column “CMAQv5.2” should read:“Nested from d01”Furthermore, the row entitled “Initial concentration (d03)” under the column “CMAQv5.2” should read:“Nested from d02”


Agelas spp. resulted in the isolation of five new bromopyrrole alkaloids, agesasines A (1) and B (2), 9-hydroxydihydrodispacamide (3), 9-hydroxydihydrooroidin (4), and 9E-keramadine (5). Their structures were elucidated on the basis of spectroscopic analyses. Agesasines A (1) and B (2) were assigned as rare bromopyrrole alkaloids lacking an aminoimidazole moiety, while 3–5 were elucidated to be linear bromopyrrole alkaloids with either aminoimidazolone, aminoimidazole, or N-methylated aminoimidazole moieties.Exploration for specialized metabolites of Okinawan marine sponges  A number of structurally unique bioactive specialized metabolites have been isolated from marine sources including sponges, algae, cnidarians, and marine microorganisms, etc. . To dateAgelas are known to be a rich source of bromopyrrole alkaloids and diterpene alkaloids that have been used as a taxonomically characteristic maker c 0.10, MeOH); UV (MeOH) λmax 275 (ε 4900) nm; 1H and 13C NMR data , [M + Na]+; HRESIMS: m/z 390.89045 [M + Na]+ .NMR data ; ESIMS: Agesasine B (2): Pale yellow amorphous solid; [α]c 0.10, MeOH); UV (MeOH) λmax 274 (ε 3100) nm; 1H and 13C NMR data , [M − H]−; HRESIMS: m/z 380.9088 [M − H]− .NMR data ; ESIMS: 9-Hydroxydihydrodispacamide (3): Pale yellow amorphous solid; [α]c 0.10, MeOH); UV (MeOH) λmax 223 (ε 3900) and 275 (3400) nm; 1H and 13C NMR data , [M − H + Na]+; HRESIMS: m/z 443.92824 [M − H + Na]+ .NMR data ; ESIMS: 9-Hydroxydihydrooroidin (4): Pale yellow amorphous solid; [α]c 0.10, MeOH); UV (MeOH) λmax 276 (ε 3900) nm; 1H and 13C NMR data , [M]+; HRESIMS: m/z 405.9510 [M]+ .NMR data ; ESIMS: E-Keramadine9 (5): Pale yellow amorphous solid; UV (MeOH) λmax 271 (ε 3300) nm; 1H and 13C NMR data (m/z 324 and 326 (1:1), [M]+; HRESIMS: m/z 324.04592 [M]+ .NMR data ; ESIMS: 1) and 9-hydroxydihydrodispacamide (3), were performed on chiral HPLC  at 35 °C with elution of MeOH/MeCN/H2O/H3PO4  to give enantiomers in the integral ratio of ca. 1:1  in each case. The separations of enantiomers were confirmed by MS analyses. Separated peaks for enantiomers of agesasine B (2) could not be obtained in any condition in this study.Optical resolutions of agesasine A  according to the following procedure. The human cancer cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS). All cells were incubated at 37 °C in a humidified atmosphere with 5% CO2–95% air. Cells were seeded at 1 × 104 cells/well in a 96-well plate and preincubated for 24 h. Test samples were dissolved in small amounts of DMSO and diluted in the appropriate culture medium . After removal of the preincubated culture medium, 100 µL of medium containing various concentrations of test compound was added and further incubated for 48 h. A cell proliferation assay was performed with the Cell Counting Kit-8  according to the manufacturer’s instruction. Briefly, the WST-8 reagent solution (10 µL) was added to each well of a 96-well microplate containing 100 µL of cells in the culture medium at various densities, and the plate was incubated for 2 h at 37 °C. Absorbance was measured at 450 nm using a microplate reader. Cisplatin was used as a positive control, whose IC50 values against HeLa, A549, and MCF7 cells were 11.7, 7.2, and 52.4 mM, respectively.New bromopyrrole alkaloids


Scientific Reports 10.1038/s41598-020-65359-5, published online 21 May 2020Correction to: The Article contains a typographical error in Table 2 where the WHO recommended limit for Hg in Soils (for agriculture) was incorrectly given as “– 0.08” ppm. The correct recommended limit should read as “0.05–0.08” ppm.


To measure myoPS, 400 ml of deuterium oxide (D2O) (70 atom %) was ingested the day prior to starting the study and m. vastus lateralis biopsies were taken before and after RT-intervention. Myofibrillar fractional synthetic rate (myoFSR) was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Pyr-IRMS). Muscle recovery parameters  were assessed daily. Results: MyoFSR PRE was 1.6 (0.2) %∙d−1 (mean (SD)). Whey protein supplementation had no effect on myoFSR (p = 0.771) or any recovery parameter (p = 0.390–0.989). Conclusions: Over an intense 7-d RT protocol, 0.33 g·kg−1·d−1 of supplemental whey protein does not enhance day-to-day measures of myoPS or postexercise recovery in resistance-trained men. Background: The aim of this study was to investigate the effect of whey protein supplementation on myofibrillar protein synthesis (myoPS) and muscle recovery over a 7-d period of intensified resistance training (RT). Methods: In a double-blind randomised parallel group design, 16 resistance-trained men aged 18 to 35 years completed a 7-d RT protocol, consisting of three lower-body RT sessions on non-consecutive days. Participants consumed a controlled diet (146 kJ·kg Over recent decades, the role that nutrition and/or exercise has on muscle protein synthesis (MPS) has been guided by the use of isotopically labelled amino acid tracers in acute lab-based studies. Thus, it is known that a single bout of resistance-exercise training (RT) stimulates a postexercise increase in mixed-muscle and myofibrillar protein synthesis (myoPS) ,2,3. Whe2O) labelling methods to measure muscle protein fractional synthetic rate (FSR) . For statistical analysis normality and homogeneity were confirmed prior to performing parametric statistical tests. Mixed-model ANOVA was used to verify group × time interaction . Independent t-tests were used to assess differences between groups. Repeated-measures ANOVA and paired t-tests were used to assess time effects on pooled data . The critical significance level was set as α = 0.05. A false-discovery rate correction was used to adjust for familywise error . The mag % CI. Fop > 0.276). Participants were weight-stable over the study protocol (± 2 kg). Meal and snack containers were inspected daily to verify adherence to the study diet. Participants consumed 98% of their required total dietary protein intake. Baseline characteristics are presented in 2O bolus ingestion, body water enrichment increased to 0.54% (0.08%), then followed a single-phase exponential decay curve (r2 = 0.993) to 0.30% (0.05%) (−1 vs. CON = 1.59 (0.19) %∙d−1, p = 0.776). Whey protein supplementation did not increase myoFSR  (−1 to 1.72 (0.24) %∙d−1, 0.11  %∙d−1, p = 0.106, ES = 0.5, n = 15).Twenty-four hours after D (0.05%) A. No difS < 0.1) B. Over tp < 0.001, ES > 1.8)  and +24 h after session 2 (day 4)  (p = 0.923) or CMJ height (p = 0.441) recovery between RT-bouts. Participants completed 8.9 (1.9) sets per session. A deficit in ISq force was observed at all time-points after the first RT session (day 2 to day 7) (S > 1.8) A and a dS = 0.7) . Whey pr−1, 94% , p < 0.001, ES = 1.3) then returned to baseline thereafter (p = 0.989). An increase in lower-body muscle soreness was reported at all time-points after session 1 (day 2 to day 7)  (p = 0.390)An increase in serum CK was observed +24 h after session 1 (day 2) (138 (87) to 215 (89) IU∙Lereafter . Whey prS > 0.9) ; however−1 (WHEY = 2.7 (0.4) μg·mg−1, CON = 2.3 (0.1) μg·mg−1). There was no supplement (p = 0.856) or RT-effect .Muscle RNA at baseline was 2.5 μg·mgIn the present study, we investigated the effect of whey protein supplementation on myoPS and indices of muscle recovery, over a 7-d intensified RT protocol. Our data show that in a cohort of young healthy resistance-trained men, daily supplementation of whey protein did not augment myoFSR or any measure of muscle recovery over multiple days of RT.2O-method, we reported that acute ingestion of 0.33 g·kg−1 of whey protein increased postexercise myoFSR, ~30 % for ~5 h, in a young healthy resistance-trained cohort [2O-tracer technique was employed to measure myoFSR, but this time measuring the effect of 0.33 g·kg−1·d−1 of supplemental whey protein over multiple days of RT. We believe the conflicting results between this study and the acute lab-based studies [In a recent study, using the Dd cohort . In the  studies ,5,6,7 ar2O-derived, measures of myoPS are predictive of longer-term physical changes to the muscle [−1⸱d−1 of 2.0 g·kg−1⸱d−1) of the total daily protein intake. We believe that of the ‘free-living’ factors, it was the participants’ relatively high dietary protein intake  [In response to RT, there is evidence to suggest that day-to-day, De muscle ,11, where muscle . To our g−1⸱d−1) ,18 that −1 (~ 20 g of protein) [−1⸱d−1 [−1⸱d−1 does not enhance RT-induced increases in fat-free mass [−1) high-quality protein supplementation, increasing total daily protein intake from 1.7 g·kg−1⸱d−1 from 2.0 g·kg−1⸱d−1, confers no further increase in myoPS. A number of acute lab-based studies have shown a dose-dependent relationship between protein ingestion and postprandial/postexercise myoFSR, plateauing at > 0.31 g·kgprotein) ,5,20. Ho [−1⸱d−1 ,18. It iree mass . Indeed,n = 15, P = 0.106, ES = 0.5). However, several studies, with similar sample sizes, have reported statistically significant increases in D2O-derived measures of myoPS in response to RT [Pooled data analysis revealed that, independent of supplements group, the RT-protocol failed to evoke a substantial increase in myoPS, not reaching the critical significance level  affected the results, thus, limiting our findings to young healthy resistance-trained men. Further research is still warranted to confirm if protein supplementation, with or without exercise, enhances day-to-day measures of myoPS in other relevant populations . Nevertheless, this study provides important information and insight into how the D−1⸱d−1 of whey protein supplementation over an intensified 7-d RT protocol fails to enhance integrative rates of myoPS or recovery in young health resistance-trained men consuming adequate amounts of dietary protein (1.7 g·kg−1⸱d−1).In conclusion, 0.33 g·kg


Scientific Reports 10.1038/s41598-019-52474-1, published online 18 November 2019Correction to: The Supplementary Information file accompanying this Article contains errors.In Supplementary Table S6A, ‘PKD1 protein-truncating mutations in study families’, the protein change for Family ID ‘5, 117’,“p.Gly3808X”should read:“p.Trp3808X”In the same table, the cDNA change for Family ID ‘213’,“c.10429-2_10429-1delCA”should read:“c.10220+2_10220+3del”In addition, in Supplementary Table S6C, ‘PKD1 non-truncating mutations in study families’, the protein change for Family ID ‘304’,“p.Gly381Arg”should read:“p.Gly381Ser”


Scientific Reports 10.1038/s41598-017-15415-4, published online 08 November 2017Correction to: In Figure 1D, the column ‘103E’ is incorrectly given as ‘N1’. In Figure 5A, the kDa for the blot GST-HAI-2 KD ‘43’ is incorrectly given as ‘55’. The correct Figure 1 and Figure 5 appear below as Figure