Palmitic acid induces GSDMD-mediated pyroptosis in periodontal ligament cells via the NF-κB pathway.

OBJECTIVE: Obesity can affect periodontal tissues and exacerbate periodontitis. Pyroptosis, a newly identified type of inflammatory cell death, is involved in the development of periodontal inflammation. The saturated fatty acid palmitic acid (PA) is elevated in obese patients. The effect of PA on pyroptosis in periodontal ligament cells (PDLCs) and its underlying mechanisms remain unknown. MATERIALS AND METHODS: Human PDLCs were isolated from healthy individuals and cultured for experiments. The effects of PA on PDLC pyroptosis and the underlying mechanisms were examined by transmission electron microscopy, quantitative real-time PCR and western blotting. RESULTS: The morphology of PDLCs in the PA group indicated pyroptotic characteristics, including swollen cells, plasma membrane rupture and changes in subcellular organelles. PA induced inflammatory responses in PDLCs, as indicated by an increase in IL-1ß in the cell culture supernatant. Furthermore, we found that the pyroptosis-related proteins caspase-1, caspase-4 and GSDMD were involved in PA-induced cell death. GSDMD and caspase-4 inhibitors alleviated pyroptotic death of PDLCs. Moreover, PA promoted NF-κB P65 phosphorylation. A NF-κB inhibitor decreased IL-1ß expression and partly rescued cell death induced by PA. CONCLUSION: PA activated the NF-κB pathway and induced the inflammatory response in PDLCs. Caspase-4/GSDMD mediated PDLC pyroptosis induced by PA.

[Diagnostic Value of Speech Acoustic Analysis in Parkinson's Disease].

Screening for and identifying patients with Parkinson's disease (PD) at an early stage and forming accurate diagnosis of PD during the course of the progression of the disease are of essential importance but still remain challenging for the clinical diagnosis and treatment of PD. One of the common clinical manifestations of PD is speech impairment, or voice impairment. Thanks to the recent advances in the field of acoustic analysis, a large number of acoustic parameters have been proposed for evaluating speech impairment quantitatively. Early identification and accurate diagnosis of PD was henceforth made possible through the application of speech acoustic analysis. Herein, we summarized the latest research findings on the application of acoustic analysis in PD diagnosis. We reported some acoustic parameters commonly used in the evaluation of voice impairment in PD patients. Then, we presented the diagnostic value of acoustic analysis in developing accurate diagnosis, early screening and differential diagnosis. Furthermore, we discussed the drawbacks and prospects of current studies, intending to enhance understanding of acoustic analysis of PD patients and its potential diagnostic values.

Emergence of 2D high-temperature nodal-line half-metal in monolayer AgN.

Nodal-line half-metals (NLHMs) are highly desirable for future spintronic devices due to their exotic quantum properties. However, the experimental realization in spin-polarized materials is nontrivial to date. Herein we perform first-principles calculations to demonstrate a 2D honeycomb, AgN, as a promising candidate of NLHMs, which is thermodynamically and dynamically stable. Band structure analysis reveals that two concentric NLs coexist centered at a Γ point near EF, accompanied by the electron and hole pockets that touch each other linearly with single-spin components. Inclusion of SOC can enrich the electronic structures of AgN, sensitive to the protection of mirror reflection symmetry: the NLHM survives if the spin is perpendicular to the Mz mirror plane, while it tunes into Wyle nodal-points by rotating spins from the out-of-plane to the in-plane direction. The characteristics of HM and NL can be well maintained on semiconducting h-BN and is immune to mechanical strains. These tunable magnetic properties render 2D AgN suitable for exotic quantum transports in nodal fermions as well as related spintronic devices.

Carbazole Alkaloids from Clausena anisum-olens: Isolation, Characterization, and Anti-HIV Evaluation.

Two new carbazole alkaloids (1,2) and six known carbazole alkaloids (3-8) were isolated from Clausena anisum-olens. Their structures were elucidated based on extensive spectroscopic analysis. All isolated compounds (1-8) were evaluated for their anti-HIV effects on virus replication in MT-4 lymphocytes infected by HIV-1NL4-3 Nanoluc-sec virus, and new carbazole alkaloid 1 exhibited anti-HIV activity with an EC50 value of 2.4 µg/mL and SI of 7.1.

Synthesis of new ent-labdane diterpene derivatives from andrographolide and evaluation on cytotoxic activities.

There are many reports for andrographolide modification regarding antitumor effects. Transformation of the five-membered lactone ring to furan aromatic ring still results in compounds with good cytotoxicity. To determine further the importance of the five-membered lactone ring and to obtain better lead compounds, we transformed the five-membered lactone ring in andrographolide. New types of ent-labdane diterpene derivatives were made, whose cytotoxic activities were measured in vitro. Preliminary SAR was summarized and two compounds, 7 and 26, with good cytotoxic activity were obtained, which have the potential to be developed into new antitumor drugs.

Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum.

BACKGROUND: Microalgae have been an emerging biofuel resource; however, the germplasm improvement has been slow due to the lack of molecular tools. Pyruvate dehydrogenase kinase (PDK) deactivates the pyruvate dehydrogenase complex (PDC) which catalyzes the oxidative decarboxylation of pyruvate. Acetyl-CoA production via PDC is important in plant tissues that are active in fatty acid synthesis. RESULTS: A 1261-bp cDNA of a putative PDK gene (PtPDK) was cloned from a diatom Phaeodactylum tricornutum, and PtPDK antisense knockdown transgenic diatoms were generated. Both PtPDK transcript abundance and enzyme activity were reduced significantly due to antisense knockdown of PtPDK. Neutral lipid content of transgenic diatom cells increased up to 82% as determined by Nile red staining, and fatty acid composition was not altered. Transgenic cells showed slightly lower growth rate but similar cell size with the wild type, hence retaining similar biomass productivity. CONCLUSIONS: This work first obtained a successful engineered diatom regulating a key gene involved in lipid metabolism. Our findings also provide powerful indications in enhancing microalgal lipid production by metabolic engineering for biofuel industry.

Functional characterization of an ACCase subunit from the diatom Phaeodactylum tricornutum expressed in Escherichia coli.

The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.

Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum.

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.

Molecular epidemiological characteristics of Streptococcus pyogenes strains involved in an outbreak of scarlet fever in China, 2011.

OBJECTIVE: To investigate molecular characterization of streptococcus pyogenes isolates involved in an outbreak of scarlet fever in China in 2011. METHODS: Seventy-four Streptococcal pyogenes involved in an outbreak of scarlet fever were isolated from pediatric patients in the areas with high incidence in China from May to August of 2011. Emm genotyping, pulsed-field gel electrophoresis (PFGE), superantigen (SAg) genes and antimicrobial susceptibility profiling were analyzed for these isolates. RESULTS: A total of 4 different emm types were identified. Emm12 was the most prevalent type which contained four predominating PFGE patterns corresponding to four different virulence and superantigen profiles. Emm12 (79.7%) and emm1 (14.9%) accounted for approximately 94% of all the isolates. The speA gene was all negative in emm12 isolates and positive in emm1 isolates. All strains were resistant to erythromycin, and 89.4% of them were resistant to erythromycin, tracycline, and clindamycin simultaneously. CONCLUSION: Several highly diversified clones with a high macrolide resistance rate comprise a predominant proportion of circulating strains, though no new emm type was found in this outbreak. The data provide a baseline for further surveillance of scarlet fever, which may contribute to the explanation of the outbreak and development of a GAS vaccine in China.

Arbuscular mycorrhizal fungi enhance active ingredients of medicinal plants: a quantitative analysis.

Medicinal plants are invaluable resources for mankind and play a crucial role in combating diseases. Arbuscular mycorrhizal fungi (AMF) are widely recognized for enhancing the production of medicinal active ingredients in medicinal plants. However, there is still a lack of comprehensive understanding regarding the quantitative effects of AMF on the accumulation of medicinal active ingredients. Here we conducted a comprehensive global analysis using 233 paired observations to investigate the impact of AMF inoculation on the accumulation of medicinal active ingredients. This study revealed that AMF inoculation significantly increased the contents of medicinal active ingredients by 27%, with a particularly notable enhancement observed in flavonoids (68%) and terpenoids (53%). Furthermore, the response of medicinal active ingredients in belowground organs (32%) to AMF was more pronounced than that in aboveground organs (18%). Notably, the AMF genus Rhizophagus exhibited the strongest effect in improving the contents of medicinal active ingredients, resulting in an increase of over 50% in both aboveground and belowground organs. Additionally, the promotion of medicinal active ingredients by AMF was attributed to improvements in physiological factors, such as chlorophyll, stomatal conductance and net photosynthetic rate. Collectively, this research substantially advanced our comprehension of the pivotal role of AMF in improving the medicinal active ingredients of plants and provided valuable insights into the potential mechanisms driving these enhancements.

Difference and clinical value of metabolites in plasma and feces of patients with alcohol-related liver cirrhosis.

BACKGROUND: Alterations in plasma and intestinal metabolites contribute to the pathogenesis and progression of alcohol-related liver cirrhosis (ALC). AIM: To explore the common and different metabolites in the plasma and feces of patients with ALC and evaluate their clinical implications. METHODS: According to the inclusion and exclusion criteria, 27 patients with ALC and 24 healthy controls (HCs) were selected, and plasma and feces samples were collected. Liver function, blood routine, and other indicators were detected with automatic biochemical and blood routine analyzers. Liquid chromatography-mass spectrometry was used to detect the plasma and feces metabolites of the two groups and the metabolomics of plasma and feces. Also, the correlation between metabolites and clinical features was analyzed. RESULTS: More than 300 common metabolites were identified in the plasma and feces of patients with ALC. Pathway analysis showed that these metabolites are enriched in bile acid and amino acid metabolic pathways. Compared to HCs, patients with ALC had a higher level of glycocholic acid (GCA) and taurocholic acid (TCA) in plasma and a lower level of deoxycholic acid (DCA) in the feces, while L-threonine, L-phenylalanine, and L-tyrosine increased simultaneously in plasma and feces. GCA, TCA, L-methionine, L-phenylalanine, and L-tyrosine in plasma were positively correlated with total bilirubin (TBil), prothrombin time (PT), and maddrey discriminant function score (MDF) and negatively correlated with cholinesterase (CHE) and albumin (ALB). The DCA in feces was negatively correlated with TBil, MDF, and PT and positively correlated with CHE and ALB. Moreover, we established a P/S BA ratio of plasma primary bile acid (GCA and TCA) to fecal secondary bile acid (DCA), which was relevant to TBil, PT, and MDF score. CONCLUSION: The enrichment of GCA, TCA, L-phenylalanine, L-tyrosine, and L-methionine in the plasma of patients with ALC and the reduction of DCA in feces were related to the severity of ALC. These metabolites may be used as indicators to evaluate the progression of alcohol-related liver cirrhosis.

Chronic intermittent hypoxia impaired collagen synthesis in mouse genioglossus via ROS accumulation: A transcriptomic analysis.

Obstructive sleep apnea (OSA) is a sleep-related breathing disorder characterized by intermittent and recurrent upper airway collapse during sleep that leads to chronic intermittent hypoxia (CIH). The genioglossus (GG) is the largest dilator muscle, which controls the upper airway and plays an important role in OSA pathology. Elucidating its genetic alterations may help identify potential targets for OSA. However, the genetic aspects of the GG in CIH mice remain unclear. Here, we have conducted an RNA sequencing (RNA-Seq) analysis to assess the differentially expressed genes (DEGs) in the GG between CIH mice and normoxia (NOR) mice. A total of 637 DEGs were identified to be dysregulated in CIH mice compared with control mice. Bioinformatics analysis showed that the DEGs were related to various physiological processes, such as the endogenous stimulus responses, cellular component organization and metabolic processes. Extracellular matrix (ECM)-receptor interaction was the top KEGG pathway in the environmental information processing category with high significance and large fold changes. From the gene weight distributions of collagen (Col)-related biological processes (BPs), we found several significant DEGs, such as Col1a1, Col1a2, Mmp2, Col3a1, Col5a1, Fmod, and Col5a2. A PPI network showed that Col1a1 was linked to ECM-receptor interactions, responses to reactive oxygen species (ROS) and Col-related BPs. It was verified in vivo and in vitro that hypoxia can induce excess ROS and reduce Col expression levels. Moreover, we found NAC can effectively scavenge ROS and restore collagen synthesis. These findings contribute to a better understanding of the mechanisms linking OSA and upper airway muscle injury and may help identify potential therapeutic targets.

Multifunctional Cd-CP for fluorescence sensing of Cr(VI), MnO4-, acetylacetone and ascorbic acid in aqueous solutions.

The development of multifunctional fluorescent chemosensors for the detection of multiple targets remains challenging but of great importance. In this paper, one novel coordination polymer (CP), denoted as [Cd2(edda)(phen)2]∙H2O (compound 1, H4edda = 5,5' (ethane-1,2-diylbis(oxy)) diisophthalic acid, phen = 1,10-phenanthroline) is successfully designed and prepared under hydrothermal conditions. Structural analysis indicates that compound 1 possesses a one-dimensional (1D) double chain structure, then self-assembles into a three-dimensional (3D) supramolecular framework via π…π interactions between phen molecules. Interestingly, compound 1 is found to be tolerant in wide range of acidic to alkaline aqueous solutions (pH = 2-13). Fluorescent spectral investigations reveal that compound 1 exhibits highly selective and sensitive fluorescence responses toward MnO4-, Cr(VI) ions, acetylacetone (acac) and ascorbic acid (AA) by fluorescence quenching in the aqueous phase. The detection limits are in the very low range, reaching µM level for the detection of MnO4-, Cr(VI) ions, nM for AA and ppm for acac detection. The distinguished multi-responsive performance suggests compound 1 to be a potential multifunctional probe. Furthermore, the possible quenching mechanisms have also been systematically investigated in this work.

3D ZnII-Based coordination polymer: Synthesis, structure and fluorescent sensing property for nitroaromatic compounds.

A water-stable ZnII-based coordination polymer (CP) with excellent photophysical behavior, namely [Zn2L(atez)(H2O)2] (compound 1; H3L = 4-(2',3'-dicarboxylphenoxy); atez = 5-aminotetrazole), was successfully prepared by the solvothermal reaction of Zn ions with a π-conjugated and semi-rigid multicarboxylate ligand H3L in the presence of N-containing linker atez. Compound 1 displays a hierarchically pillared three-dimensional (3D) (3,4,5)-connected (4·62) (42·64) (43·64·83) net which is based on two-dimensional (2D) multicarboxylate- ZnII layers strutted by the atez ligands. Sensing investigations of compound 1 reveal that this material can selectively and sensitively detect nitroaromatic compounds in water suspension through fluorescence quenching effect. In particular, it is worth noting that it shows highly specific detection of nitrobenzene (NB) and 2,4,6-trinitrophenol (TNP) with remarkable quenching constants (KSV = 7.5 × 104 M-1 for NB and KSV = 1.9 × 105 M-1 for TNP) and low limit of detection (LOD = 0.93 µM for NB and LOD = 0.36 µM for TNP). Investigations reveal that the probable mechanisms for such sensing processes are the concurrent presence of fluorescence resonance energy transfer (FRET) as well as photoinduced electron transfer (PET) between the CP and nitroaromatic molecules. This work not only offers an effective route to improve the application of fluorescent CPs but also provide one novel probable fluorescence probe for nitroaromatic compounds.

Interferon-beta inhibits human glioma stem cell growth by modulating immune response and cell cycle related signaling pathways.

Malignant Glioma is characterized by strong self-renewal potential and immature differentiation potential. The main reason is that malignant glioma holds key cluster cells, glioma stem cells (GSCs). GSCs contribute to tumorigenesis, tumor progression, recurrence, and treatment resistance. Interferon-beta (IFN-ß) is well known for its anti-proliferative efficacy in diverse cancers. IFN-ß also displayed potent antitumor effects in malignant glioma. IFN-ß affect both GSCs and Neural stem cells (NSCs) in the treatment of gliomas. However, the functional comparison, similar or different effects of IFN-ß on GSCs and NSCs are rarely reported. Here, we studied the similarities and differences of the responses to IFN-ß between human GSCs and normal NSCs. We found that IFN-ß preferentially inhibited GSCs over NSCs. The cell body and nucleus size of GSCs increased after IFN-ß treatment, and the genomic analysis revealed the enrichment of the upregulated immune response, cell adhesion genes and down regulated cell cycle, ribosome pathways. Several typical cyclin genes, including cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin B2 (CCNB2), and cyclin D1 (CCND1), were significantly downregulated in GSCs after IFN-ß stimulation. We also found that continuous IFN-ß stimulation after passage further enhanced the inhibitory effect. Our study revealed how genetic diversity resulted in differential effects in response to IFN-ß treatment. These results may contribute to improve the applications of IFN-ß in anti-cancer immunotherapy. In addition, these results may also help to design more effective pharmacological strategies to target cancer stem cells while protecting normal neural stem cells.

Improving Biotransformation Efficiency of Arthrobacter simplex by Enhancement of Cell Stress Tolerance and Enzyme Activity.

Arthrobacter simplex exhibits excellent Δ1-dehydrogenation ability, but the acquisition of the desirable strain is limited due to lacking of comprehensive genetic manipulation. Herein, a promoter collection for fine-tuning gene expression was achieved. Next, the expression level was enhanced and directed evolution of the global transcriptional factor (IrrE) was applied to enhance cell viability in systems containing more substrate and ethanol, thus contributing to higher production. IrrE promotes a stronger antioxidant defense system, more energy generation, and changed signal transduction. Using a stronger promoter, the enzyme activities were boosted but their positive effects on biotransformation performance were inferior to cell stress tolerance when exposed to challenging systems. Finally, an optimal strain was created by collectively reinforcing cell stress tolerance and catalytic enzyme activity, achieving a yield 261.8% higher relative to the initial situation. Our study provided effective methods for steroid-transforming strains with high efficiency.

A Fast and Efficient Approach to Obtaining High-Purity Glioma Stem Cell Culture.

Glioma is the most common and malignant primary brain tumor. Patients with malignant glioma usually have a poor prognosis due to drug resistance and disease relapse. Cancer stem cells contribute to glioma initiation, progression, resistance, and relapse. Hence, quick identification and efficient understanding of glioma stem cells (GSCs) are of profound importance for therapeutic strategies and outcomes. Ideally, therapeutic approaches will only kill cancer stem cells without harming normal neural stem cells (NSCs) that can inhibit GSCs and are often beneficial. It is key to identify the differences between cancer stem cells and normal NSCs. However, reports detailing an efficient and uniform protocol are scarce, as are comparisons between normal neural and cancer stem cells. Here, we compared different protocols and developed a fast and efficient approach to obtaining high-purity glioma stem cell by tracking observation and optimizing culture conditions. We examined the proliferative and differentiative properties confirming the identities of the GSCs with relevant markers such as Ki67, SRY-box containing gene 2, an intermediate filament protein member nestin, glial fibrillary acidic protein, and s100 calcium-binding protein (s100-beta). Finally, we identified distinct expression differences between GSCs and normal NSCs including cyclin-dependent kinase 4 and tumor protein p53. This study comprehensively describes the features of GSCs, their properties, and regulatory genes with expression differences between them and normal stem cells. Effective approaches to quickly obtaining high-quality GSCs from patients should have the potential to not only help understand the diseases and the resistances but also enable target drug screening and personalized medicine for brain tumor treatment.

Emergence of a spin-valley Dirac semimetal in a strained group-VA monolayer.

The combination of Dirac and Valley physics in one single-layer system is a very interesting topic and has received widespread attention in materials science and condensed matter physics. Using density-functional theoretical calculations, we predict that a two-dimensional (2D) cyanided group-VA monolayer, MAs(CN)2 (M = Sb, Bi), can turn into the spin-valley Dirac point (svDP) state under external strains. In sharp contrast to the symmetry protected 2D Dirac semimetal (DSM), the Dirac Fermions in svDP materials are spin non-degenerate due to strong spin-splitting under SOC. Remarkably, the Dirac fermions in inequivalent valleys can host opposite Berry curvature and spin moment, leading to the Dirac spin-valley Hall effect with dissipationless transport. We also find that the svDP of MAs(CN)2 is a critical state of topological phase transition between the trivial and nontrivial states. An effective tight-binding model is used to unveil the physics of svDP and topological phase transition under strain. These results will provide a route towards the integration of spin-valley indexes in 2D Dirac materials and design multipurpose and controllable devices in valleytronics.

Aging Induced p53/p21 in Genioglossus Muscle Stem Cells and Enhanced Upper Airway Injury.

Aging of population brings related social problems, such as muscle attenuation and regeneration barriers with increased aging. Muscle repair and regeneration depend on muscle stem cells (MuSCs). Obstructive sleep apnea (OSA) rises in the aging population. OSA leads to hypoxia and upper airway muscle injury. However, little is known about the effect of increasing age and hypoxia to the upper airway muscle. The genioglossus (GG) is the major dilator muscle to keep the upper airway open. Here, we reported that muscle fiber and MuSC function declined with aging in GG. Increasing age also decreased the migration and proliferation of GG MuSCs. p53 and p21 were high expressions both in muscle tissue and in GG MuSCs. We further found that hypoxia inhibited GG MuSC proliferation and decreased myogenic differentiation. Then, hypoxia enhanced the inhibition effect of aging to proliferation and differentiation. Finally, we investigated that hypoxia and aging interact to form a vicious circle with upregulation of p53 and p21. This vicious hypoxia plus aging damage accelerated upper airway muscle injury. Aging and hypoxia are the major damage elements in OSA patients, and we propose that the damage mechanism of hypoxia and aging in GG MuSCs will help to improve upper airway muscle regeneration.

Evaluation of the long-term stability of micro-screws under different loading protocols: a systematic review.

The aim of this systematic review was to investigate the association between the different factors of loading protocols and the long-term stability of micro-screws from biomechanical and histological viewpoints. Searches were performed on PubMed, Embase, Cochrane Library, Wanfang and CNKI databases for animal experiments comparing loading protocols and the long-term stability of micro-screws. Among 1011 detected papers, 16 studies met the eligibility criteria and were selected for analysis. Most studies showed medium methodological quality for evaluation of micro-screws' long-term stability. Five studies reported that loading would not destroy the long-term stability of micro-screws. Three studies indicated that low-intensity immediate loading or a 3-week minimal healing time was acceptable. Two studies reported that the loading magnitude was a controversial issue with regard to the micro-screws' long-term stability. Two studies suggested that counterclockwise loading could decrease the long-term stability of micro-screws. In conclusion, immediate loading below 100g force, healing time greater than 3 weeks, regular loading below 200g force and a clockwise direction of force supported the long-term stability of micro-screws. Further studies relating to the combination of varying loading conditions will be needed.