Structural basis of inhibition of the human SGLT2-MAP17 glucose transporter.

Human sodium-glucose cotransporter 2 (hSGLT2) mediates the reabsorption of the majority of filtrated glucose in the kidney1. Pharmacological inhibition of hSGLT2 by oral small-molecule inhibitors, such as empagliflozin, leads to enhanced excretion of glucose and is widely used in the clinic to manage blood glucose levels for the treatment of type 2 diabetes1. Here we determined the cryogenic electron microscopy structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state to an overall resolution of 2.95 Å. Our structure shows eukaryotic SGLT-specific structural features. MAP17 interacts with transmembrane helix 13 of hSGLT2. Empagliflozin occupies both the sugar-substrate-binding site and the external vestibule to lock hSGLT2 in an outward-open conformation, thus inhibiting the transport cycle. Our work provides a framework for understanding the mechanism of SLC5A family glucose transporters and also develops a foundation for the future rational design and optimization of new inhibitors targeting these transporters.

An aldehyde-crosslinking mitochondrial probe for STED imaging in fixed cells.

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential-dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions.

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain.

Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

Enhancing Thermoelectric Performance in P-Type Sb2Te3-Based Compounds Through Nb─Ag Co-Doping with Donor-Like Effect.

P-type Sb2Te3 has been recognized as a potential thermoelectric material for applications in low-medium temperature ranges. However, its inherent high carrier concentration and lattice thermal conductivity led to a relatively low ZT value, particularly around room temperature. This study addresses these limitations by leveraging high-energy ball milling and rapid hot-pressing techniques to substantially enhance the Seebeck coefficient and power factor of Sb2Te3, yielding a remarkable ZT value of 0.55 at 323 K due to the donor-like effect. Furthermore, the incorporation of Nb─Ag co-doping increases hole concentration, effectively suppressing intrinsic excitations ≈548 K while maintaining the favorable power factor. Simultaneously, the lattice thermal conductivity can be significantly reduced upon doping. As a result, the ZT values of Sb2Te3-based materials attain an impressive range of 0.5-0.6 at 323 K, representing an almost 100% improvement compared to previous research endeavors. Finally, the ZT value of Sb1.97Nb0.03Ag0.005Te3 escalates to 0.92 at 548 K with a record average ZT value (ZTavg) of 0.75 within the temperature range of 323-573 K. These achievements hold promising implications for advancing the viability of V-VI commercialized materials for low-medium temperature application.

The Remarkable Role of Indium in Synergistically Optimizing Carrier Concentration and Phase Distribution of AgCuTe-Based Materials.

Carrier regulation has proven to be an effective approach for optimizing the thermoelectric performance of materials. One common method to adjust the carrier concentration is through element doping. In the case of AgCuTe-based materials, it tends to form with cation vacancies, resulting in a high hole concentration and complex phase composition at low temperatures, which also hinders material stability. However, this also offers additional opportunities to manipulate the carrier concentration. In this study, the improved performance of AgCuTe through indium doping is reported, which leads to a reduction in hole concentration. In combination with a significant increase in the effective mass of the carriers, the enhanced Seebeck coefficient is also realized. Particularly, a notable improvement in power factor is observed in the hexagonal phase near room temperature. Furthermore, a lower electron thermal conductivity is achieved, contributing to an average figure of merit value of ≈1.21 (between 523 and 723 K). Additionally, the presence of indium inhibits the formation of the second phase and ensures a homogeneous phase distribution, which reduces the instability arising from phase transition. This work significantly enhances the potential of AgCuTe-based materials for low to medium-temperature applications.

Formic acid sandwich method is well-suited for filamentous fungi identification and improves turn around time using Zybio EXS2600 mass spectrometry.

OBJECTIVES: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is extensively employed for the identification of filamentous fungi on MALDI Biotyper (Bruker Daltonics) and Vitek MS (biomerieux), but the performance of fungi identification on new EXS2600 (Zybio) is still unknow. Our study aims to evaluate the new EXS2600 system's (Zybio) ability to rapidly identify filamentous fungi and determine its effect on turnaround time (TAT) in our laboratory. METHODS: We tested 117 filamentous fungi using two pretreatment methods: the formic acid sandwich (FA-sandwich) and a commercial mold extraction kit (MEK, Zybio). All isolates were confirmed via sequence analysis. Laboratory data were extracted from our laboratory information system over two 9-month periods: pre-EXS (April to December 2022) and post-EXS (April to December 2023), respectively. RESULTS: The total correct identification (at the species, genus, or complex/group level) rate of fungi was high, FA-sandwich (95.73%, 112/117), followed by MEK (94.02%, 110/117). Excluding 6 isolates not in the database, species-level identification accuracy was 92.79% (103/111) for FA-sandwich and 91.89% (102/111) for MEK; genus-level accuracy was 97.29% (108/111) and 96.39% (107/111), respectively. Both methods attained a 100% correct identification rate for Aspergillus, Lichtheimia, Rhizopus Mucor and Talaromyces species, and were able to differentiate between Fusarium verticillioides and Fusarium proliferatum within the Fusarium fujikuroi species complex. Notably, high confidence was observed in the species-level identification of uncommon fungi such as Trichothecium roseum and Geotrichum candidum. The TAT for all positive cultures decreased from pre EXS2600 to post (108.379 VS 102.438, P < 0.05), and the TAT for tissue decreased most (451.538 VS 222.304, P < 0.001). CONCLUSIONS: The FA-sandwich method is more efficient and accurate for identifying filamentous fungi with EXS2600 than the MEK. Our study firstly evaluated the performance of fungi identification on EXS2600 and showed it is suitable for clinical microbiology laboratories use.

Transient Stimulated Raman Excited Fluorescence Spectroscopy.

The pursuit of better sensitivity has always been one of the central themes in Raman spectroscopy. Recently, all-far-field single-molecule Raman spectroscopy has been demonstrated by a novel hybrid spectroscopy that couples Raman scattering with fluorescence emission. However, such frequency-domain spectroscopy lacks efficient hyperspectral excitation methods and encounters intrinsic strong fluorescence backgrounds from electronic transitions, hindering its applications in advanced Raman spectroscopy and microscopy. Here we report the ultrafast time-domain spectroscopy counterpart named transient stimulated Raman excited fluorescence (T-SREF): excited by two successive broadband femtosecond pulse pairs (i.e., the pump and Stokes pulses) with time-delay scanning, strong vibrational wave packet interference is revealed on the time-domain fluorescence trace, resulting in background-free spectra of the corresponding Raman modes after the Fourier transform. T-SREF achieves background-free Raman spectra of electronic-coupled vibrational modes with sensitivity up to the level of a few molecules, which paves the way for supermultiplexed fluorescence detection and molecular dynamics sensing.

General Strategy To Improve the Photon Budget of Thiol-Conjugated Cyanine Dyes.

Maleimide-cysteine chemistry has been a routine practice for the site-specific labeling of fluorophores to proteins since the 1950s. This approach, however, cannot bring out the best photon budget of fluorophores. Here, we systematically measured the Cyanine3/5 dye conjugates via maleimide-thiol and amide linkages by counting the total emitted photons at the single-molecule level. While brightness and signal-to-noise ratios do not change significantly, dyes with thioether linkages exhibit more severe photobleaching than amide linkers. We then screened modern arylation-type bioconjugation strategies to alleviate this damage. Labeling thiols with phenyloxadiazole (POD) methyl sulfone, p-chloronitrobenzene, and fluorobenzene probes gave rise to electron-deficient aryl thioethers, effectively increasing the total emitted photons by 1.5-3 fold. Among the linkers, POD maintains labeling efficiency and specificity that are comparable to maleimide. Such an increase has proved to be universal among bulk and single-molecule assays, with or without triplet-state quenchers and oxygen scavengers, and on conformationally unrestricted or restricted cyanines. We demonstrated that cyanine-POD conjugates are general and superior fluorophores for thiol labeling in single-molecule FRET measurements of biomolecular conformational dynamics and in two-color STED nanoscopy using site-selectively labeled nanobodies. This work sheds light on the photobleaching mechanism of cyanines under single-molecule imaging while highlighting the interplay between the protein microenvironment, bioconjugation chemistry, and fluorophore photochemistry.

Diazo-carboxyl Click Derivatization Enables Sensitive Analysis of Carboxylic Acid Metabolites in Biosamples.

Carboxylic acids are central metabolites in bioenergetics, signal transduction, and post-translation protein regulation. However, the quantitative analysis of carboxylic acids as an indispensable part of metabolomics is prohibitively challenging, particularly in trace amounts of biosamples. Here we report a diazo-carboxyl/hydroxylamine-ketone double click derivatization method for the sensitive analysis of hydrophilic, low-molecular-weight carboxylic acids. In general, our method renders a 5- to 2000-fold higher response in mass spectrometry along with improved chromatographic separation. With this method, we presented the near-single-cell analysis of carboxylic acid metabolites in 10 mouse egg cells before and after fertilization. Malate, fumarate, and ß-hydroxybutyrate were found to decrease after fertilization. We also monitored the isotope labeling kinetics of carboxylic acids inside adherent cells cultured in 96-well plates during drug treatment. Finally, we applied this method to plasma or serum samples (5 µL) collected from mice and humans under pathological and physiological conditions. The double click derivatization method paves a way toward single-cell metabolomics and bedside diagnostics.

Evaluation of the Autof ms1000 mass spectrometry for rapid clinical identification of filamentous fungi.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized microbial identification. However, there is a lack of data on its performance in identifying filamentous fungi. The objective of our study was to evaluate the accuracy of the Autof ms1000 mass spectrometry for identifying filamentous fungi in the clinical microbiology laboratory. RESULTS: Among 106 samples tested using the Autof ms1000 system, 101 (95.28%) were identified at the genus or species level, and 81 (76.41%) were accurately identified at the species level. Additionally, we developed a new rapid formic acid extraction method with simple pretreatment for filamentous fungi that saved time and provided accurate results. CONCLUSIONS: The Autof ms1000 mass spectrometer proved to be a valuable tool for identifying filamentous fungi. However, upgrading the database is recommended for correctly identifying rare strains.

Super-multiplex vibrational imaging.

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Two-phase orthodontic treatment of a patient with a low-angle skeletal class II malocclusion: a 7-year follow-up.

Low-angle skeletal class II malocclusions are often observed with sagittal and vertical developmental abnormalities of the mandible. Two-phase orthodontic treatment of functional orthopedic therapy combined with fixed correction is one of the most common methods to treat of skeletal class II malocclusions. This case report describes the two-phase orthodontic treatment of a patient with severe low-angle skeletal class II malocclusion. A Twin Block orthodontic appliance was used to improve mandibular growth, and the adjustment of the occlusal relationship using a fixed appliance after functional therapy. After treatment, a significant improvement was observed in the patient's facial appearance and occlusal relationship. Additionally, a 7-year follow-up confirmed the stability of the treatment results. Although a vertical facial growth direction is difficult to control, the Twin Block orthodontic appliance in adolescents might effectively improve the difference in the sagittal growth of the mandible. Whilst the growth pattern could not be fully controlled, the treatment significantly improved the patient's facial profile and occlusion.

Emerging Trends in Fluorescence Bioimaging of Divalent Metal Cations Using Small-Molecule Indicators.

Visualization of cation dynamics inside a living system represent a major breakthrough at the crossroad of chemistry and cellular physiology. Since the inception of BAPTA-based cellular calcium indicators in the 1980s, generations of chemical and genetically encoded ion indicators spanning the visible spectrum have been developed. In this article, we bring up three emerging concepts in this field: 1. red-shifting cation indicators towards far-red and near-infrared (NIR) channels; 2. directing the indicators to various subcellular localizations; 3. lowering the phototoxicity of indicators for long term recording. These initiatives collectively echo the advocate of 4D cellular physiology, where biological processes within living systems can be panoramically unveiled under 3D, long-term, and multi-channel imaging with unprecedented spatial and temporal resolution. This outlook poses exciting challenges and opportunities for chemists to upgrade the toolkit of fluorescent indicators as key enablers for a new era of imageomics.

Evaluation of a Cryptococcus capsular polysaccharide detection FungiXpert LFA (lateral flow assay) for the rapid diagnosis of Cryptococcosis.

Cryptococcus is an opportunistic pathogenic fungus and is the major cause of fungal meningitis. The cryptococcal antigen (CrAg) lateral flow assay (LFA) is an immunochromatographic test system that has simplified diagnosis as a point-of-care test. In this study, we evaluated the diagnostic performance of Cryptococcal capsular polysaccharide detection FungiXpert (Genobio Pharmaceutical, Tianjin, China) using serum and cerebrospinal fluid (CSF) samples for the diagnosis of cryptococcosis and investigated the cross-reaction of the assays to pathogenic fungi and bacterium by comparing it to the U.S. Food and Drug Administration (US FDA)-approved IMMY CrAg LFA. Eighty CSF and 119 serum/plasma samples from 158 patients were retrospectively collected to test for qualitative or semi-quantitative detection of CrAg. Cross-reaction of the assays was tested using 28 fungi and 1 bacterium. Compared to IMMY CrAg LFA, the FungiXpert LFA demonstrated 99.1% sensitivity and 98.9% specificity in the qualitative test. In the 96 semi-quantitative CrAg assay results, 39 (40.6%) test titers of FungiXpert LFA were 1-2 dilutions higher than those of IMMY CrAg LFA. The Intraclass Correlation Coefficient of the Semi-quantitative results of CrAg titer tests via the two assays was 0.976. Similar to IMMY CrAg LFA, FungiXpert LFA showed cross-reactivity with Trichosporon asahii. Compared with the IMMY CrAg LFA, the FungiXpert LFA showed an equal, yet, excellent performance. However, it is important to note that these two assays have potential cross-reactivity to T. asahii when diagnosing patients. FungiXpert LFA is a rapid screening method for the effective and practical diagnosis and treatment of cryptococcosis. LAY SUMMARY: The FungiXpert LFA was developed to diagnose fungal meningitis caused by Cryptococcus yeasts, by using serum or cerebrospinal fluid. It was compared to an existing lateral flow assay (LFA). The FungiXpert LFA performed well in qualitative and semi-quantitative tests.

Cutaneous Phaeohyphomycosis of the Right Hand Caused by Exophiala jeanselmei: A Case Report and Literature Review.

Exophiala spp. is increasingly reported as a pathogen causing the cutaneous, subcutaneous or invasive infection. In this report, we present a case of cutaneous phaeohyphomycosis due to E. jeanselmei on the right hand of a farmer, who suffered from this disease three years ago which had not been definitely diagnosed until he was admitted to our hospital. In our hospital, a potential fungal pathogen was observed by histopathological examination, and then was recovered and identified as E. jeanselmei by sequencing its internal transcribed spacer region. After 4 weeks of antifungal treatment, his hand recovered very well. To investigate the in vitro susceptibility of E. jeanselmei isolates to antifungal agents and compare the characteristics of their related infections among immunocompetent and immunocompromised patients, we reviewed 84 cases published in PubMed database between 1980 and 2020.

Third-Generation Covalent TMP-Tag for Fast Labeling and Multiplexed Imaging of Cellular Proteins.

Self-labeling protein tags can introduce advanced molecular motifs to specific cellular proteins. Here we introduce the third-generation covalent TMP-tag (TMP-tag3) and showcase its comparison with HaloTag and SNAP-tag. TMP-tag3 is based on a proximity-induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)-acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP-tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18-kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal N-C termini of eDHFR also enable facile insertion into various protein loops. TMP-tag3, HaloTag, and SNAP-tag are orthogonal to each other, collectively forming a toolbox for multiplexed live-cell imaging of cellular proteins under fluorescence nanoscopy.

Emergomyces orientalis Emergomycosis Diagnosed by Metagenomic Next-Generation Sequencing.

Emergomyces is a newly described dimorphic fungus genus; it may cause fatal infections in immunocompromised patients, but diagnosis is often delayed. We report a case of disseminated emergomycosis caused by the novel species Emergomyces orientalis in a kidney transplant recipient from Tibet. Infection was diagnosed early by metagenomic next-generation sequencing.

Molecular epidemiology and antifungal susceptibilities of Cryptococcus species isolates from HIV and non-HIV patients in Southwest China.

To investigated the molecular epidemiology and in vitro antifungal susceptibility of Cryptococcus isolates from West China Hospital from HIV and non-HIV patients between 2009 and 2015. A total of 132 C. neoformans and C. gattii were subjected to antifungal susceptibility testing by E-test method. Among the 132 isolates, 42 C. neoformans and C. gattii were analyzed by mating type and URA5-RFLP. A total of 113 C. neoformans and C. gattii were subjected to multi-locus sequence typing (MLST). MLST results revealed that ST5 was the major molecular type. The wild-type (WT) phenotype was seen in 91.5-100% of C. neoformans isolates for amphotericin B, 5-flucytosine, fluconazole, and voriconazole. However, 72.3% (94/130) of C. neoformans isolates were non-wild-type (non-WT) to itraconazole by E-test method. In the sixth study year, the geometric mean, MIC50 and MIC90 of fluconazole were the highest (P < 0.001). Among 132 patients. 52 were coinfected with HIV and 80 were HIV-negative. Isolates from HIV and non-HIV patients showed no differences in susceptibility to amphotericin B (P = 0.544), 5-flucytosine (P = 0.063), fluconazole (P = 0.570), voriconazole (P = 0.542), and itraconazole (P = 0.787). Our study showed that Cryptococcus in southwest China showed a low degree of genetic diversity. The increased MIC values of fluconazole are noted. Cryptococcus isolates from HIV and non-HIV patients have shown no differences in susceptibility to five antifungal agents.

A Meta-analysis and Systematic Review Comparing the Effectiveness of Traditional and Virtual Surgical Planning for Orthognathic Surgery: Based on Randomized Clinical Trials.

PURPOSE: To explore the advantages of virtual surgical planning (VSP) and traditional surgical planning (TSP) to determine whether the current VSP technique is superior to the TSP technique for orthognathic surgery. METHODS: An electronic search was carried out in the CENTRAL, PubMed, and Embase databases to identify randomized clinical trials (RCTs) that compared the VSP and TSP techniques regarding their surgical accuracy for hard tissue, prediction precision for soft tissue, required time for planning and surgery, cost and patient-reported outcomes. RESULTS: Eight articles from 5 RCTs, involving 199 patients, were identified. The findings showed that the VSP and TSP techniques were similar in surgical accuracy for hard tissue in the sagittal plane, although the VSP technique was significantly more accurate in certain reference areas, especially in the anterior area of the maxilla. Both the VSP and TSP techniques had significantly better surgical accuracy for the maxilla than for the mandible. The VSP technique showed clinically significantly greater precision for soft tissue prediction in the sagittal plane. Patients who were treated via the VSP technique presented a more symmetrical frontal view, regardless of whether hard or soft tissue was involved. The VSP technique required more time for software planning, but it showed an advantage in time savings when considering the entire preoperative process. Accompanied by the use of an accurate computer-aided splint, the VSP technique could effectively reduce the operative time. Apart from the initial financial investment of software and hardware, the total cost of the VSP technique was similar to that of the TSP technique. Patients who were treated via the VSP or TSP technique showed similar improvements in quality-of-life. CONCLUSIONS: Currently, the VSP technique has become a good alternative to the TSP technique for orthognathic surgery, especially regarding frontal-esthetic considerations. Studies reporting indicators with good representativeness and sensitivity using an identical comparative method are recommended.