TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during Development and in Aging.

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.

PARP5A and RNF146 phase separation restrains RIPK1-dependent necroptosis.

Phase separation is a vital mechanism that mediates the formation of biomolecular condensates and their functions. Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3, and MLKL downstream of TNFR1 and has been implicated in mediating many human diseases. However, whether necroptosis is regulated by phase separation is not yet known. Here, we show that upon the induction of necroptosis and recruitment by the adaptor protein TAX1BP1, PARP5A and its binding partner RNF146 form liquid-like condensates by multivalent interactions to perform poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 in mouse embryonic fibroblasts. We show that PARdU predominantly occurs on the K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis. Our data demonstrate that PARdU on K376 of mouse RIPK1 provides an alternative cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146.

Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.

Modulating TRADD to restore cellular homeostasis and inhibit apoptosis.

Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.

Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death.

TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.

Necroptosis in development and diseases.

Necroptosis, a form of regulated necrotic cell death mediated by RIPK1 (receptor-interacting protein kinase 1) kinase activity, RIPK3, and MLKL (mixed-lineage kinase domain-like pseudokinase), can be activated under apoptosis-deficient conditions. Modulating the activation of RIPK1 by ubiquitination and phosphorylation is critical to control both necroptosis and apoptosis. Mutant mice with kinase-dead RIPK1 or RIPK3 and MLKL deficiency show no detrimental phenotype in regard to development and adult homeostasis. However, necroptosis and apoptosis can be activated in response to various mutations that result in the abortion of the defective embryos and human inflammatory and neurodegenerative pathologies. RIPK1 inhibition represents a key therapeutic strategy for treatment of diseases where blocking both necroptosis and apoptosis can be beneficial.

HOIP modulates the stability of GPx4 by linear ubiquitination.

LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death.

USP25 regulates Wnt signaling by controlling the stability of tankyrases.

Aberrant activation of the Wnt signaling pathway plays an important role in human cancer development. Wnt signaling is negatively regulated by Axin, a scaffolding protein that controls a rate-limiting step in the destruction of ß-catenin, the central activator of the Wnt pathway. In Wnt-stimulated cells, Axin is rapidly modified by tankyrase-mediated poly(ADP-ribosyl)ation, which promotes the proteolysis of Axin and consequent stabilization of ß-catenin. Thus, regulation of the levels and activity of tankyrases is mechanistically important in controlling Wnt signaling. Here, we identify ubiquitin-specific protease 25 (USP25) as a positive regulator of Wnt/ß-catenin signaling. We found that USP25 directly interacted with tankyrases to promote their deubiquitination and stabilization. We demonstrated that USP25 deficiency could promote the degradation of tankyrases and consequent stabilization of Axin to antagonize Wnt signaling. We further characterized the interaction between TNKS1 and USP25 by X-ray crystal structure determination. Our results provide important new insights into the molecular mechanism that regulates the turnover of tankyrases and the possibility of targeting the stability of tankyrases by antagonizing their interaction with USP25 to modulate the Wnt/ß-catenin pathway.

SPATA2 regulates the activation of RIPK1 by modulating linear ubiquitination.

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.

The nature of crystal facet effect of TiO2-supported Pd/Pt catalysts on selective hydrogenation of cinnamaldehyde: electron transfer process promoted by interfacial oxygen species.

Supported noble metal nanocatalysts typically exhibit strong crystal plane dependent catalytic behavior, but their working mechanism is still unclear. Herein, using anatase TiO2 with well-exposed crystal facets of {101}, {100} and {001} as a prototype support, Pd- and Pt-based supported TiO2 nanocatalysts (TiO2-Pd and TiO2-Pt) were prepared by chemical reduction with NaBH4 as reducer, and they showed a distinct metal-dependent crystal facet effect in the selective hydrogenation of cinamaldehyde (CAL). For Pd-based nanocatalysts, most Pd species on the {100} plane of TiO2 are present in the oxidized form with positive charges and unexpectedly show higher reactivity than the Pd species in the zero-valence state on the {101} and {001} planes. On the contrary, Pt species on all three crystal planes of TiO2 show zero-valence state, with relatively low conversion, but much better selectivity for hydrogenation of a CO bond than Pd-based catalysts. Well-designed experiments manipulating the stability and type of surface oxygen species confirmed that the essence of the crystal facet effect of the catalyst support actually creates a unique nanoconfined interface at the molecular level to construct a surface p-band intermediate state (PBIS), which provides a new alternative channel for surface electron transfer and consequently accelerates the reaction kinetics.

Surgical outcomes of robotic-assisted percutaneous fixation for thoracolumbar fractures in patients with ankylosing spondylitis.

BACKGROUND: Spinal fractures in patients with ankylosing spondylitis (AS) mainly present as instability, involving all three columns of the spine, and surgical intervention is often considered necessary. However, in AS patients, the significant alterations in bony structure and anatomy result in a lack of identifiable landmarks, which increases the difficulty of pedicle screw implantation. Therefore, we present the clinical outcomes of robotic-assisted percutaneous fixation for thoracolumbar fractures in patients with AS. METHODS: A retrospective review was conducted on a series of 12 patients diagnosed with AS. All patients sustained thoracolumbar fractures between October 2018 and October 2022 and underwent posterior robotic-assisted percutaneous fixation procedures. Outcomes of interest included operative time, intra-operative blood loss, complications, duration of hospital stay and fracture union. The clinical outcomes were assessed using the visual analogue scale (VAS) and Oswestry Disability Index (ODI). To investigate the achieved operative correction, pre- and postoperative radiographs in the lateral plane were analyzed by measuring the Cobb angle. RESULTS: The 12 patients had a mean age of 62.8 ± 13.0 years and a mean follow-up duration of 32.7 ± 18.9 months. Mean hospital stay duration was 15 ± 8.0 days. The mean operative time was 119.6 ± 32.2 min, and the median blood loss was 50 (50, 250) ml. The VAS value improved from 6.8 ± 0.9 preoperatively to 1.3 ± 1.0 at the final follow-up (P < 0.05). The ODI value improved from 83.6 ± 6.1% preoperatively to 11.8 ± 6.6% at the latest follow-up (P < 0.05). The average Cobb angle changed from 15.2 ± 11.0 pre-operatively to 8.3 ± 7.1 at final follow-up (P < 0.05). Bone healing was consistently achieved, with an average healing time of 6 (5.3, 7.0) months. Of the 108 screws implanted, 2 (1.9%) were improperly positioned. One patient experienced delayed nerve injury after the operation, but the nerve function returned to normal upon discharge. CONCLUSION: Posterior robotic-assisted percutaneous internal fixation can be used as an ideal surgical treatment for thoracolumbar fractures in AS patients. However, while robot-assisted pedicle screw placement can enhance the accuracy of pedicle screw insertion, it should not be relied upon solely.

USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1.

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 is known to inhibit UPS. Here we show that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1. Furthermore, we show that activation of USP14 by Akt-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Our study suggests that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. We propose that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases.

Molecular Wiring of Electrocatalytic Nitrate reduction to Ammonia and Water Oxidation by Iron-Coordinated Macroporous Conductive Networks.

Developing stable electrocatalysts with accessible isolated sites is desirable but highly challenging due to metal agglomeration and low surface stability of host materials. Here we report a general approach for synthesis of single-site Fe electrocatalysts by integrating a solvated Fe complex in conductive macroporous organic networks through redox-active coordination linkages. Electrochemical activation of the electrode exposes high-density coordinately unsaturated Fe sites for efficient adsorption and conversion of reaction substrates such as NO3 - and H2O. Using the electrode with isolated active Fe sites, electrocatalytic NO3 - reduction and H2O oxidation can be coupled in a single cell to produce NH3 and O2 at Faradaic efficiencies of 97 % and 100 %, respectively. The electrode exhibits excellent robustness in electrocatalysis for 200 hours with small decrease in catalytic efficiencies. Both the maximized Fe-site efficiency and the microscopic localization effect of the conductive organic matrix contribute to the high catalytic performances, which provides new understandings in tuning the efficiencies of metal catalysts for high-performance electrocatalytic cells.

Molecular Self-Assembly in Conductive Covalent Networks for Selective Nitrate Electroreduction to Ammonia.

Electrochemical nitrate (NO3-) reduction in aqueous media provides a useful approach for ammonia (NH3) synthesis. While efforts are focused on developing catalysts, the local microenvironment surrounding the catalyst centers is of great importance for controlling electrocatalytic performance. Here, we demonstrate that a self-assembled molecular iron catalyst integrated in a free-standing conductive hydrogel is capable of selective production of NH3 from NO3- at efficiencies approaching unity. With the electrocatalytic hydrogel, the NH3 selectivity is consistently high under a range of negative biases, which results from the hydrophobicity increase of the polycarbazole-based electrode substrate. In mildly acidic media, proton reduction is suppressed by electro-dewetting of the hydrogel electrode, enhancing the selectivity of NO3- reduction. The electrocatalytic hydrogel is capable of continuous production of NH3 for at least 100 h with NH3 selectivity of ∼89 to 98% at high current densities. Our results highlight the role of constructing an internal hydrophobic surface for electrocatalysts in controlling selectivity in aqueous media.

Atmospheric Mercury Isotope Shifts in Response to Mercury Emissions from Underground Coal Fires.

Pollutant emissions from coal fires have caused serious concerns in major coal-producing countries. Great efforts have been devoted to suppressing them in China, notably at the notorious Wuda Coalfield in Inner Mongolia. Recent surveys revealed that while fires in this coalfield have been nearly extinguished near the surface, they persist underground. However, the impacts of Hg volatilized from underground coal fires remain unclear. Here, we measured concentrations and isotope compositions of atmospheric Hg in both gaseous and particulate phases at an urban site near the Wuda Coalfield. The atmospheric Hg displayed strong seasonality in terms of both Hg concentrations (5-7-fold higher in fall than in winter) and isotope compositions. Combining characteristic isotope compositions of potential Hg sources and air mass trajectories, we conclude that underground coal fires were still emitting large amounts of Hg into the atmosphere that have been transported to the adjacent urban area in the prevailing downwind direction. The other local anthropogenic Hg emissions were only evident in the urban atmosphere when the arriving air masses did not pass directly through the coalfield. Our study demonstrates that atmospheric Hg isotope measurement is a useful tool for detecting concealed underground coal fires.

Structural water molecules dominated p band intermediate states as a unified model for the origin on the photoluminescence emission of noble metal nanoclusters: from monolayer protected clusters to cage confined nanoclusters.

In the past several decades, noble metal nanoclusters (NMNCs) have been developed as an emerging class of luminescent materials due to their superior photo-stability and biocompatibility, but their luminous quantum yield is relatively low and the physical origin of the bright photoluminescence (PL) of NMNCs remain elusive, which limited their practical application. As the well-defined structure and composition of NMNCs have been determined, in this mini-review, the effect of each component (metal core, ligand shell and interfacial water) on their PL properties and corresponded working mechanism were comprehensively introduced, and a model that structural water molecules dominated p band intermediate state was proposed to give a unified understanding on the PL mechanism of NMNCs and a further perspective to the future developments of NMNCs by revisiting the development of our studies on the PL mechanism of NMNCs in the past decade.

Mechanism of Cr(VI) removal by efficient Cr(VI)-resistant Bacillus mobilis CR3.

Cr(VI) is a hazardous environmental pollutant that poses significant risks to ecosystems and human health. We successfully isolated a novel strain of Bacillus mobilis, strain CR3, from Cr(VI)-contaminated soil. Strain CR3 showed 86.70% removal capacity at 200 mg/L Cr(VI), and a good Cr(VI) removal capacity at different pH, temperature, coexisting ions, and electron donor conditions. Different concentrations of Cr(VI) affected the activity of CR3 cells and the removal rate of Cr(VI), and approximately 3.46% of total Cr was immobilized at the end of the reaction. The combination of SEM-EDS and TEM-EDS analysis showed that Cr accumulated both on the cell surface and inside the cells after treatment with Cr(VI). XPS analysis showed that both Cr(III) and Cr(VI) were present on the cell surface, and FTIR results indicated that the presence of Cr on the cell surface was mainly related to functional groups, such as O-H, phosphate, and -COOH. The removal of Cr(VI) was mainly achieved through bioreduction, which primarily occurred outside the cell. Metabolomics analysis revealed the upregulation of five metabolites, including phenol and L-carnosine, was closely associated with Cr(VI) reduction, heavy metal chelation, and detoxification mechanisms. In addition, numerous metabolites were linked to cellular homeostasis exhibited differential expression. Cr(VI) exerted inhibitory effects on the division rate and influenced critical pathways, including energy metabolism, nucleotide metabolism, and amino acid synthesis and catabolism. These findings reveal the molecular mechanism of Cr(VI) removal by strain CR3 and provide valuable insights to guide the remediation of Cr(VI)-contaminated sites.

Dynamic Pt-OH-·H2O-Ag species mediate coupled electron and proton transfer for catalytic hydride reduction of 4-nitrophenol at the confined nanoscale interface.

Generally, the catalytic transformation of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) at heterogeneous metal surfaces follows a Langmuir-Hinshelwood (L-H) mechanism when sodium borohydride (NaBH4) is used as the sacrificial reductant. Herein, with Pt-Ag bimetallic nanoparticles confined in dendritic mesoporous silica nanospheres (DMSNs) as a model catalyst, we demonstrated that the conversion of 4-NP did not pass through the direct hydrogen transfer route with the hydride equivalents being supplied by borohydride via the bimolecular L-H mechanism, since Fourier transform infrared (FTIR) spectroscopy with the use of isotopically labeled reactants (NaBD4 and D2O) showed that the final product of 4-AP was composed of protons (or deuterons) that originated from the solvent water (or heavy water). Combined characterization by X-ray photoelectron spectroscopy (XPS), 1H nuclear magnetic resonance (NMR) and the optical excitation and photoluminescence spectrum evidenced that the surface hydrous hydroxide complex bound to the metal surface (also called structural water molecules, SWs), due to the space overlap of p orbitals of two O atoms in SWs, could form an ensemble of dynamic interface transient states, which provided the alternative electron and proton transfer channels for selective transformation of 4-NP. The cationic Pt species in the Ag-Pt bimetallic catalyst mainly acts as a dynamic adsorption center to temporally anchor SWs and related reactants, and not as the active site for hydrogen activation.

Efficient immobilization behavior and mechanism investigation of Pb(II) by Aspergillus tubingensis.

OBJECTIVES: To understand the mechanism of Pb(II) immobilized by Pb(II)-tolerant microbes. RESULTS: Aspergillus tubingensis isolated from the lead-zine mine was investigated through surface morphology observation and multiple experimental analysis in order to elucidate the Pb(II) biosorption and immobilization behavior. The maximum Pb(II) uptake capacity of A. tubingensis was about 828.8 mg L-1. Fourier transform-infrared spectra and environmental scanning electron microscope indicated that a large number of functional groups (carboxyl, phosphoryl and sulfydryl, etc.) participated in Pb(II) binding on the cell surface. Raman and X-ray diffraction, field emission high-resolution transmission electron microscopy and X-ray absorption fine structure investigation revealed that the Pb(II) loaded on the surface of the fungus could be transformed into PbCO3 and PbS nanocrystals. Meanwhile, Pb(II) transported into the cell would be oxidized to form lead oxide minerals (Pb2O3.333) over time. CONCLUSIONS: This study has important implications for an in-depth understanding of Pb(II) removal by A. tubingensis and provides guidance for remediating lead-polluted environment using microorganisms.

Steering CO2 electroreduction toward ethanol production by a surface-bound Ru polypyridyl carbene catalyst on N-doped porous carbon.

Electrochemical reduction of CO2 to multicarbon products is a significant challenge, especially for molecular complexes. We report here CO2 reduction to multicarbon products based on a Ru(II) polypyridyl carbene complex that is immobilized on an N-doped porous carbon (RuPC/NPC) electrode. The catalyst utilizes the synergistic effects of the Ru(II) polypyridyl carbene complex and the NPC interface to steer CO2 reduction toward C2 production at low overpotentials. In 0.5 M KHCO3/CO2 aqueous solutions, Faradaic efficiencies of 31.0 to 38.4% have been obtained for C2 production at -0.87 to -1.07 V (vs. normal hydrogen electrode) with 21.0 to 27.5% for ethanol and 7.1 to 12.5% for acetate. Syngas is also produced with adjustable H2/CO mole ratios of 2.0 to 2.9. The RuPC/NPC electrocatalyst maintains its activity during 3-h CO2-reduction periods.